Leucine and nicotinic acid reduces lipid levels

ABSTRACT

Compositions, methods, and kits useful for treating hyperlipidemic conditions are provided herein. Such compositions can contain synergizing amounts of nicotinic acid, nicotinamide riboside and/or nicotinic acid metabolites in combination with leucine and/or a leucine metabolite, with or without resveratrol.

CROSS REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/800,363, filed on Mar. 15, 2013, which is entirely incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Metabolic disorders, such as hyperlipidemia and obesity, and the relatedimpact on health and mortality presents a significant burden to publichealth. For instance, obesity, clinically defined as a body mass indexof over 30 kg/m2, is estimated to affect 35.7% of the U.S. adultpopulation. In the U.S., obesity is estimated to cause roughly110,000-365,000 deaths per year. Obesity can result in hyperlipidemia,characterized by an excess of lipids, including cholesterol, cholesterolesters, phospholipids, and triglycerides, in the bloodstream, and canfurther result in diabetes, vascular disease, cancer, renal disease,infectious diseases, external causes, intentional self-harm, nervoussystem disorders, and chronic pulmonary disease (N Engl J Med 2011;364:829-841). Metabolic syndrome, in which subjects present with centralobesity and at least two other metabolic disorders (such as highcholesterol, high blood pressure, or diabetes), is estimated to affect25% of the U.S. population.

Hyperlipidemia, one of the symptoms of obesity and other conditions, canbe treated with various medications, including nicotinic acid. Nicotinicacid is a form of vitamin B3 (niacin). When taken in high doses (1-3g/day), nicotinic acid can treat hyperlipidemia, as it can lower totallipid, LDL, cholesterol, triglycerides, and lipoprotein, or raise HDLlipoprotein in the bloodstream. It can also reduce atheroscleroticplaque progression and coronary heart disease morbidity and mortality.

However, nicotinic acid can have a significant side-effect and hence canbe generally poorly tolerated. One significant side-effect can be severecutaneous vasodilation and flushing responses, and is consequentlyinfrequently prescribed despite well documented safety and efficacy(Carlson L A. Nicotinic acid: the broad-spectrum lipid drug. A 50thanniversary review. J Int Med 2005; 258:94-114). While side effects aresomewhat attenuated in sustained (SR) and extended (ER) releasepreparations, the side effects persist sufficiently to limit drug use.Therefore, there exists a pressing need to decrease the side-effects ofthe nicotinic acid without decreasing its therapeutic effects.

SUMMARY OF THE INVENTION

The subject application provides compositions, methods, and kits fortreating hyperlipidemia in a subject. The compositions, methods, andkits can include leucine and/or leucine metabolites in combination withnicotinic acid. The present invention addresses the negative sideeffects of treating subjects with high doses of nicotinic acid.

The subject compositions can be administered orally or through otherroutes such as intravenous administration. Compositions for oraladministration can include pills, tablets, capsules, and the like.

In one aspect, the current invention provides compositions comprising atleast about 250 mg of leucine and/or about 25 mg of one or more leucinemetabolites; and at least about 1 mg of nicotinic acid and/ornicotinamide riboside and/or one or more nicotinic acid metabolites.

In another aspect of the invention, compositions are provided thatcomprise at least about 250 mg of leucine and/or about 25 mg of one ormore leucine metabolites; and an amount of nicotinic acid and/ornicotinamide riboside and/or one or more nicotinic acid metabolites,wherein the composition is substantially free of each of alanine,glycine, glutamic acid, and proline.

In yet another aspect, the current invention also provides compositionscomprising at least about 250 mg of leucine and/or about 25 mg of one ormore leucine metabolites; and an amount of nicotinic acid and/ornicotinamide riboside and/or nicotinic acid metabolites, wherein theamount of nicotinic acid and/or nicotinic acid metabolites isinsufficient to reduce lipid content in the absence of component (a).

In still yet another aspect of the invention, compositions are providedthat comprise an amount of leucine and/or one or more leucinemetabolites; and an amount of nicotinic acid and/or nicotinamideriboside and/or one or more nicotinic acid metabolites, wherein thecomposition is administered to a subject in need thereof, furtherwherein the composition causes a reduced degree of cutaneousvasodilation in the subject administered as compared to a dose ofnicotinic acid alone that has the same effectiveness as the compositionin lowering lipid content in the subject.

In another aspect, current invention further provides compositionscomprising leucine and/or one or more leucine metabolites; and at leastabout 1 mg of nicotinic acid and/or nicotinamide riboside and/or one ormore nicotinic acid metabolites, wherein the molar ratio of component(a) to (b) in said composition is greater than about 20.

In some embodiments, the composition described herein comprises at leastabout 500 mg of leucine and/or at least about 200 mg of the one or moreleucine metabolites. In some embodiments, the composition can compriseat least about 250 mg of leucine and/or about 25 mg of one or moreleucine metabolites. In some embodiments, the amount of leucine and/orone or more leucine metabolites is less than about 1 g. In someembodiments, the amount of leucine and/or one or more leucinemetabolites is less than 3 g.

The compositions as disclosed herein can be substantially free ofnicotinamide. In some embodiments, the composition can be substantiallyfree of nicotinic acid metabolites. In another embodiment, thecomposition can be substantially free of each of nicotinyl CoA,nicotinuric acid, nicotinate mononucleotide, nicotinate adeninedinucleotide, and nicotinamide adenine dinucleotide. In someembodiments, the nicotinic acid and/or nicotinamide riboside and/or oneor more nicotinic acid metabolites is nicotinic acid.

The composition as disclosed herein can be substantially free of leucinemetabolites. In some embodiments, the leucine and/or one or more leucinemetabolites is leucine.

In some cases, the compositions described herein can include an amountof nicotinic acid and/or nicotinamide riboside and/or one or morenicotinic acid metabolites that can be less than about 1 g. In somecases, the amount of nicotinic acid and/or nicotinamide riboside and/orone or more nicotinic acid metabolites can be less than about 250 mg. Insome embodiments, the amount of nicotinic acid and/or nicotinamideriboside and/or one or more nicotinic acid metabolites can be betweenabout 1-100 mg. In still some embodiments, the amount of nicotinic acidand/or nicotinamide riboside and/or one or more nicotinic acidmetabolites can be at least about 1 mg.

In some embodiments, the composition described herein can include anamount of nicotinic acid and/or nicotinamide riboside and/or one or morenicotinic acid metabolites that is capable of achieving a serum level ofthe nicotinic acid and/or nicotinamide riboside and/or one or morenicotinic acid metabolites that is less than about 100 nM. In anotheraspect of the invention, the amount of nicotinic acid and/ornicotinamide riboside and/or one or more nicotinic acid metabolites canbe capable of achieving a serum level of the nicotinic acid and/ornicotinamide riboside and/or one or more nicotinic acid metabolites thatis about 10 nM. In yet another aspect, the amount of nicotinic acidand/or nicotinamide riboside and/or one or more nicotinic acidmetabolites can be capable of achieving a serum level of the nicotinicacid and/or nicotinamide riboside and/or one or more nicotinic acidmetabolites that is between about 1-100 nM.

In some cases, the composition disclosed herein can be effective inlowering triglyceride level, total cholesterol or LDL level in thesubject by at least about 5%. In some cases, the amount of component (a)and (b) in the composition can synergistically reduce lipid content insaid subject when administered to the subject. In some embodiments, thecomponent (a) and component (b) in the composition can synergisticallyenhance a decrease in weight gain of the subject, an increase in fatoxidation of the subject, or an increase in activation of Sirt1 in thesubject.

In some embodiments, the amount of nicotinic acid and/or nicotinamideriboside and/or nicotinic acid metabolites in the compositions describedherein can be insufficient to reduce lipid content in the absence of theleucine and/or one or more leucine metabolites.

In some cases, the composition disclosed herein can be contained in afoodstuff.

In some embodiments, the subject compositions can comprise a portion ofthe leucine and/or one or more leucine metabolites that is in a freeform. In some embodiments, a portion of the leucine and/or one or moreleucine metabolites can be in a salt form.

In some aspect of the invention, the composition can further compriseresveratrol.

In some cases, the composition can be formulated for oraladministration. In some cases, the composition can be a tablet, acapsule, a pill, a granule, an emulsion, a gel, a plurality of beadsencapsulated in a capsule, a powder, a suspension, a liquid, asemi-liquid, a semi-solid, a syrup, a slurry or a chewable form.

In some cases, the component (a) and component (b) comprised in thecomposition as described herein can be separately packaged. In someembodiments, the component (a) and component (b) can be mixed.

The composition as described herein can be substantially free of eachamino acid selected from the group consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine,histidine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, valine, isoleucine and tyrosine. In some cases,the composition can be substantially free of each free amino acidselected from the group consisting of alanine, arginine, asparagine,aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, valine, isoleucine and tyrosine. In some cases, thecomposition can contain less than about 0.1% of each free amino acidselected from the group consisting of alanine, arginine, asparagine,aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, valine, isoleucine and tyrosine. In some embodiments, thecomposition can contain less than about 10% of non-leucine amino acids.

In another aspect, the one or more leucine metabolites that can becomprised in the compositions as described herein can be selected fromthe group consisting of keto-isocaproic acid (KIC),alpha-hydroxy-isocaproic acid, and HMB. In some embodiments, thecomposition disclosed herein can not contain nicotinamide.

In yet another aspect, the composition disclosed herein can furthercomprise one or more therapeutic agents that are capable of loweringlipid accumulation. In some embodiments, the one or more therapeuticagents that can be comprised in the compositions described herein can beselected from the group consisting of HMG-CoA inhibitor, fibrate, bileacid sequestrant, ezetimibe, lomitapide, phytosterols, CETP antagonist,orlistat, and any combination thereof.

In some embodiments, the molar ratio of component (a) to (b) in thecomposition described herein can be greater than about 20.

In some embodiments, the composition described herein can be formulatedin a unit dosage form.

In another aspect, current invention also provides kits that comprise amulti-day supply of unit dosages of the composition described herein andinstructions directing the administration of said multi-day supply overa period of multiple days.

In yet another aspect, current invention further provides methods oflowering total cholesterol level in a subject in need thereof thatcomprise administering to said subject the composition disclosed hereinto effect the total cholesterol level in the subject. In someembodiments, current invention also provides methods of lowering totallipid content in a subject in need thereof that comprise administeringto said subject the composition disclosed herein to effect the totallipid content in the subject.

In one aspect, current invention also provides methods of lowering totalcholesterol level in a subject in need thereof that compriseadministering to said subject a dose of a composition comprising leucineand/or one or more leucine metabolites and an amount of nicotinic acidand/or nicotinic acid metabolites to effect the total cholesterol levelin the subject.

In some embodiments, the amount of nicotinic acid and/or nicotinamideriboside and/or nicotinic acid metabolites that can be used in themethods disclosed herein can be less than about 250 mg. In someembodiments, the amount of nicotinic acid and/or nicotinamide ribosideand/or nicotinic acid metabolites that can be used in the methodsdisclosed herein can be less than about 100 mg. In some embodiments, theamount of nicotinic acid and/or nicotinamide riboside and/or nicotinicacid metabolites that can be used in the methods described herein can beless than about 25 mg. In some embodiments, the amount of nicotinic acidand/or nicotinamide riboside and/or nicotinic acid metabolites that canbe used in the methods described herein can be less than about 10 mg. Insome embodiments, the dose of the composition that can be used in themethods disclosed herein can be a unit dose.

In another aspect of the invention, methods of reducing a side effect ofnicotinic acid and/or nicotinamide riboside and/or one or more nicotinicacid metabolites are disclosed, wherein the side effect can becharacterized by an increase in cutaneous vasodilation in a subjectadministered with nicotinic acid and/or nicotinamide riboside and/or theone or more nicotinic acid metabolites, and the methods compriseadministering a composition comprising an effective amount of leucineand/or one or more leucine metabolites to said subject that can beadministered with nicotinic acid and/or the one or more nicotinic acidmetabolites.

In some embodiments, the method as described herein can involveadministering the composition orally.

In some cases, the effective amount or leucine and/or leucinematabolites that are used in the methods described herein can comprisesat least about 500 mg of leucine and/or at least about 200 mg of the oneor more leucine metabolites. In some embodiments, the effective amountof the leucine and/or leucine metabolites can comprise at least about250 mg of leucine and/or at least about 25 mg of the one or more leucinemetabolites.

In some embodiments, the methods disclosed herein can involve nicotinicacid and/or nicotinamide riboside and/or one or more nicotinic acidmetabolites that is in a sub-therapeutic amount if administered alone.In some embodiments, the nicotinic acid and/or nicotinamide ribosideand/or one or more nicotinic acid metabolites used in the methodsdescribed herein can be in an amount that is less than about 1 g. Insome embodiments, the nicotinic acid and/or nicotinamide riboside and/orone or more nicotinic acid metabolites used in the methods disclosedherein can be in an amount that is less than about 250 mg. In someembodiments, the amount of nicotinic acid and or nicotinamide ribosideand/or one or more nicotinic acid metabolites that is used in themethods described herein can be between about 1-100 mg.

In some cases, the methods described herein can use an amount ofnicotinic acid and or nicotinamide riboside and/or one or more nicotinicacid metabolites that is capable of achieving a serum level of nicotinicacid and/or nicotinamide riboside and/or one or more nicotinic acidmetabolites that is between about 1-100 nM.

In yet another aspect of the invention, methods of reducingatherosclerotic plaque size in a subject in need thereof are provided.The methods comprise administering to said subject a dose of acomposition comprising leucine and/or one or more leucine metabolitesand an amount of nicotinic acid and/or nicotinic acid metabolites toeffect the total atherosclerotic plaque size in the subject.

In some embodiments, the amount of nicotinic acid and/or nicotinamideriboside and/or nicotinic acid metabolites that can be used in themethods described herein can be less than about 250 mg. In someembodiments, the amount of nicotinic acid and/or nicotinamide ribosideand/or nicotinic acid metabolites can be between about 1-100 mg. In someembodiments, the amount of nicotinic acid and/or nicotinamide ribosideand/or nicotinic acid metabolites can be less than about 25 mg.

In some cases, the methods described herein can involve administeringthe composition to the subject for at least about 1 year.

In some embodiments, the dose of the composition used in the methodsdescribed herein can be a unit dose.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates the chemical structures of nicotinic acid andnicotinamide riboside.

FIG. 2 illustrates the effects of nicotinic acid and leucine, and/orresveratrol on Sirt1 activation in C2C12 myotubes. NA refers tonicotinic acid; Leu refers to leucine; R refers to resveratrol. *p<0.05;**p=0.0001. Data expressed as % change from control value.

FIG. 3 illustrates the effects of nicotinic acid and leucine, and/orresveratrol on P-AMPK/AMPK ratio in 3T3-L1 adipocytes. NA refers tonicotinic acid; Leu refers to leucine; R refers to resveratrol. *p<0.01.Data expressed as % change from control value.

FIG. 4 illustrates the effects of leucine (0.5 mM)/nicotinic acid (10nM) on lipid levels in C. elegans (*p=0.012). NA refers to nicotinicacid; Leu refers to leucine.

FIG. 5 illustrates the effects of four weeks treatment with Leucine(Leu, 24 g/kg diet), Leu (24 g/kg diet)+nicotinic acid (NA, 50 mg/kgdiet), Leu (24 g/kg diet)+NA (250 mg/kg diet) and NA (1,000 mg/kg diet)added to a Western Diet (WD) on plasma total cholesterol in LDL receptorknockout mice.

FIG. 6 illustrates the effects of four weeks treatment with Leucine(Leu, 24 g/kg diet), Leu (24 g/kg diet)+nicotinic acid (NA, 50 mg/kgdiet), Leu (24 g/kg diet)+NA (250 mg/kg diet) and NA (1,000 mg/kg diet)added to a Western Diet (WD) on plasma cholesterol esters in LDLreceptor knockout mice.

FIG. 7 illustrates the effects of four weeks treatment with Leucine(Leu, 24 g/kg diet), Leu (24 g/kg diet)+nicotinic acid (NA, 50 mg/kgdiet), Leu (24 g/kg diet)+NA (250 mg/kg diet) and NA (1,000 mg/kg diet)added to a Western Diet (WD) on plasma triglycerides in LDL receptorknockout mice.

FIG. 8 illustrates the effects of nicotinic acid and leucine on thelifespan of C. elegans. NA refers to nicotinic acid; Leu refers toleucine. *p<0.0001. Data expressed as % survival over time.

DETAILED DESCRIPTION OF THE INVENTION

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, up to 10%, up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated the term “about” meaning within an acceptable errorrange for the particular value should be assumed.

As used herein, the term “subject” or “individual” includes mammals.Non-limiting examples of mammals include humans and mice, includingtransgenic and non-transgenic mice. The methods described herein can beuseful in both human therapeutics, pre-clinical, and veterinaryapplications. In some embodiments, the subject is a mammal, and in someembodiments, the subject is human. Other mammals include, and are notlimited to, apes, chimpanzees, orangutans, monkeys; domesticated animals(pets) such as dogs, cats, guinea pigs, hamsters, mice, rats, rabbits,and ferrets; domesticated farm animals such as cows, buffalo, bison,horses, donkey, swine, sheep, and goats; or exotic animals typicallyfound in zoos, such as bear, lions, tigers, panthers, elephants,hippopotamus, rhinoceros, giraffes, antelopes, sloth, gazelles, zebras,wildebeests, prairie dogs, koala bears, kangaroo, pandas, giant pandas,hyena, seals, sea lions, and elephant seals.

The terms “administer”, “administered”, “administers” and“administering” are defined as the providing a composition to a subjectvia a route known in the art, including but not limited to intravenous,intraarterial, oral, parenteral, buccal, topical, transdermal, rectal,intramuscular, subcutaneous, intraosseous, transmucosal, orintraperitoneal routes of administration. In certain embodiments of thesubject application, oral routes of administering a composition can bepreferred.

As used herein, “agent” or “biologically active agent” refers to abiological, pharmaceutical, or chemical compound or other moiety.Non-limiting examples include simple or complex organic or inorganicmolecule, a peptide, a protein, a peptide nucleic acid (PNA), anoligonucleotide (including e.g., aptomer and polynucleotides), anantibody, an antibody derivative, antibody fragment, a vitaminderivative, a carbohydrate, a toxin, or a chemotherapeutic compound.Various compounds can be synthesized, for example, small molecules andoligomers (e.g., oligopeptides and oligonucleotides), and syntheticorganic compounds based on various core structures. In addition, variousnatural sources can provide compounds for screening, such as plant oranimal extracts, and the like. A skilled artisan can readily recognizethat there is no limit as to the structural nature of the agents of thepresent invention.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound described herein that is sufficient toaffect the intended application including but not limited to disease orcondition treatment, as defined below. The therapeutically effectiveamount can vary depending upon the intended application (in vitro or invivo), or the subject and disease condition being treated, e.g., theweight and age of the subject, the severity of the disease condition,the manner of administration and the like, which can readily bedetermined by one of ordinary skill in the art. The term also applies toa dose that will induce a particular response in target cells, e.g.,reduction of proliferation or down regulation of activity of a targetprotein. The specific dose will vary depending on the particularcompounds chosen, the dosing regimen to be followed, whether it isadministered in combination with other compounds, timing ofadministration, the tissue to which it is administered, and the physicaldelivery system in which it is carried.

The term “energy metabolism,” as used herein, refers to thetransformation of energy that accompanies biochemical reactions in thebody, including cellular metabolism and mitochondrial biogenesis. Energymetabolism can be quantified using the various measurements describedherein, for example and without limitations, weight-loss, fat-loss,insulin sensitivity, fatty acid oxidation, glucose utilization,triglyceride content, Sirt 1 expression level, AMPK expression level,oxidative stress, and mitochondrial biomass.

The term “isolated”, as applied to the subject components, for example aPDE 5 inhibitor, including but not limited to nicotinic acid and/ornicotinamide riboside and/or one or more nicotinic acid metabolites,leucine and leucine metabolites (such as HMB), and resveratrol, refersto a preparation of the substance devoid of at least some of the othercomponents that can also be present where the substance or a similarsubstance naturally occurs or is initially obtained from. Thus, forexample, an isolated substance can be prepared by using a purificationtechnique to enrich it from a source mixture. Enrichment can be measuredon an absolute basis, such as weight per volume of solution, or it canbe measured in relation to a second, potentially interfering substancepresent in the source mixture. Increasing enrichment of the embodimentsof this invention are increasingly more preferred. Thus, for example, a2-fold enrichment is preferred, 10-fold enrichment is more preferred,100-fold enrichment is more preferred, 1000-fold enrichment is even morepreferred. A substance can also be provided in an isolated state by aprocess of artificial assembly, such as by chemical synthesis.

A “sub-therapeutic amount” of an agent, an activator or a therapy is anamount less than the effective amount of that agent, activator ortherapy for an intended application, but when combined with an effectiveor sub-therapeutic amount of another agent or therapy can produce adesired result, due to, for example, synergy in the resultingefficacious effects, and/or reduced side effects.

A “synergistic” or “synergizing” effect can be such that the one or moreeffects of the combination compositions are greater than the one or moreeffects of each component alone, or they can be greater than the sum ofthe one or more effects of each component alone. The synergistic effectcan be about, or greater than about 10, 20, 30, 50, 75, 100, 110, 120,150, 200, 250, 350, or 500% or even more than the effect on a subjectwith one of the components alone, or the additive effects of each of thecomponents when administered individually. The effect can be any of themeasurable effects described herein.

The term “substantially free”, as used herein, refers to compositionsthat have less than about 10%, less than about 5%, less than about 1%,less than about 0.5%, less than 0.1% or even less of a specifiedcomponent. For example a composition that is substantially free ofnon-branched chain amino acids can have less than about 1% of thenon-branched chain amino acid lysine. The percentage can be determinedas a percent of the total composition or a percent of a subset of thecomposition. For example, a composition that is substantially free ofnon-branched chain amino acids can have less than 1% of the non-branchedchain amino acids as a percent of the total composition, or as a percentof the amino acids in the composition. The percentages can be mass,molar, or volume percentages.

The terms “clinical significance” or “clinically significant” indicatebehaviors and symptoms that are considered to be outside the range ofnormal, and are marked by distress and impairment of daily functioning.For example, a clinically significant cutaneous vasodilation would be alevel sufficient to elicit patient complaint regarding discomfortsecondary to acute vasodilatation, including flushing, itching and/ortingling. Levels of cutaneous vasodilation can also be measured by anymethods known in the medical art, such as the methods includinglaser-Doppler flowmeter that are disclosed in Saumet J. L. et al.,“Non-invasive measurement of skin blood flow: comparison betweenplethysmography, laser-Doppler flowmeter and heat thermal clearancemethod” Int. J. Microcirc. Clin. Exp. 1986; 5:73-83. A clinicallysignificant level of cutaneous vasodilation can also be a level that isstatistically significant. A clinically significant level of cutaneousvasodilation can also be a level that is not statistically significant.

The terms “lipid content” or “lipid level” refer to the content or levelof lipid or lipoprotein molecules measured inside of a subject. It canbe the concentration of the lipid molecules in a circulatingbloodstream, or a total quantity of body fat. The lipid or lipoproteinmolecules can include triglyceride, cholesterol, LDL, or HDL.

Compositions

The subject compositions comprise a combination of (i) leucine and/orone or more leucine metabolites, and (ii) nicotinic acid and/ornicotinamide riboside and/or one or more nicotinic acid metabolites. Thecomposition can be used to treat hyperlipidemia. The compositions canfurther comprise resveratrol or one or more therapeutic agents that iscapable of lowering lipid level. The combination of these components canbe useful for lowering lipid content, lowering total cholesterol level,lowering LDL level, lowering triglyceride level or increasing HDL level.In some embodiments, the components are formulated to provide asynergistic effect, including but not limited to further reduction ofthe fat content or reduction in dosing amounts leading to reduced sideeffects to the subject. The combination can be particularly effective inlowering the lipid content while causing a reduced degree of cutaneousvasodilation in a subject as compared to a dose of nicotinic acid alonethat has the same effectiveness as the composition in lowering lipidcontent. The amount of nicotinic acid and/or nicotinamide ribosideand/or one or more nicotinic acid metabolites in the composition can bea sub-therapeutic amount in the absence of the leucine and/or one ormore leucine metabolites.

In one embodiment, the subject composition comprises leucine and/or oneor more leucine metabolites; and nicotinic acid and/or nicotinamideriboside and/or nicotinic acid metabolites, wherein the compositioncomprises at least about 250 mg of leucine and/or at least about 25 mgof the one or more leucine metabolites, and further wherein thecomposition comprises at least about 1 mg of nicotinic acid and/ornicotinamide riboside and/or one or more nicotinic acid metabolites.

In another embodiment, the subject composition comprises leucine and/orone or more leucine metabolites; and nicotinic acid and/or nicotinamideriboside and/or nicotinic acid metabolites, wherein the compositioncomprises at least about 250 mg of leucine and/or at least about 10, 20,25, 30, 35, 40, 45, 50, 55, 60 mg of the one or more leucinemetabolites, and further wherein the composition is substantially freeof each of the amino acids including but are not limited to: alanine,glycine, glutamic acid and proline.

In yet another embodiment, the subject composition comprises leucineand/or one or more leucine metabolites; and an amount of nicotinic acidand/or nicotinamide riboside and/or nicotinic acid metabolites, whereinthe composition comprises at least about 250 mg of leucine and/or atleast about 25 mg of the one or more leucine metabolites, and furtherwherein the amount of nicotinic acid and/or nicotinamide riboside and/ornicotinic acid metabolites is insufficient to demonstrate a therapeuticeffect such as reducing lipid content in the absence of the leucineand/or one or more leucine metabolites. In some embodiments, the amountof the nicotinic acid and/or nicotinamide riboside and/or one or morenicotinic acid metabolites is sub-therapeutic when administered withoutleucine and/or one or more leucine metabolites.

In still yet another embodiment, the subject composition comprisesleucine and/or one or more leucine metabolites; and nicotinic acidand/or nicotinamide riboside and/or nicotinic acid metabolites, whereinthe composition is effective in lowering lipid content in a subject inneed thereof while causing a reduced degree of cutaneous vasodilation inthe subject as compared to a dose of nicotinic acid alone that has thesame effectiveness as the composition in lowering lipid content. In someembodiments, the composition is effective in lowering lipid content in asubject in need thereof without causing a clinically significantcutaneous vasodilation.

In another embodiment, the subject composition comprises (a) leucineand/or one or more leucine metabolites; and (b) nicotinic acid and/ornicotinamide riboside and/or nicotinic acid metabolites, wherein themass ratio of (a) to (b) is at least about 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, or 100, and wherein the compositioncomprises at least about 1 mg of the nicotinic acid and/or nicotinamideriboside and/or nicotinic acid metabolites. As described herein, adosing of at least about 1 mg of nicotinic acid and/or nicotinamideriboside and/or nicotinic acid metabolites can provide a sub-therapeuticdosing that can be effective when combined with a sufficient mass ratioof leucine or leucine metabolite.

In some embodiments, the subject composition comprises (a) leucineand/or one or more leucine; and (b) nicotinic acid and/or nicotinamideriboside and/or one or more nicotinic acid metabolites, whereincomponent (a) and component (b) have synergistic effects. Thesynergistic effects can be synergistically enhances a decrease in weightgain of the subject, a decrease in lipid content, a decrease in LDLlevel, an increase in HDL level, a decrease in cholesterol level, adecrease in triglyceride level, an increase in fat oxidation of thesubject, or an increase in activation of Sirt1 in the subject.

In one aspect of the invention, the subject composition comprises (a)leucine and/or one or more leucine; and (b) nicotinic acid and/ornicotinamide riboside and/or one or more nicotinic acid metabolites. Thecomposition can further comprise at least about 0.01, 0.05, 0.1, 0.5, or1 μg of resveratrol.

In yet another embodiment, the subject composition comprises (a) leucineand/or one or more leucine metabolites; and (b) nicotinic acid and/ornicotinamide riboside and/or nicotinic acid metabolites, wherein (b) arepresent in an amount effective to achieve a circulating level of about1-100 nM of nicotinic acid and/or nicotinamide riboside and/or nicotinicacid metabolites in a subject. In some embodiments, the circulatinglevel of nicotinic acid and/or nicotinamide riboside and/or nicotinicacid metabolites is 10 nM. These targeted circulating levels correspondto treatment concentrations described herein (see Examples), which wereshown to provide beneficial effects on hyperlipidemic conditions in asubject.

Nicotinic Acid, Nicotinamide Riboside and Nicotinic Acid Metabolites

The invention provides for compositions that include nicotinic acidand/or nicotinamide riboside and/or nicotinic acid metabolites. Thenicotinic acid and/or nicotinamide riboside and/or nicotinic acidmetabolites can be used in free form. The term “free,” as used herein inreference to a component, indicates that the component is notincorporated into a larger molecular complex. In some embodiments, thenicotinic acid can be comprised in niacin. The nicotinic acid and/ornicotinamide riboside and/or nicotinic acid metabolites can be in a saltform.

In some embodiments, the compositions can be substantially free ofnicotinamide and/or nicotinamide metabolites. The nicotinamide and/ornicotinamide metabolites can counteract the effects of nicotinic acid ornicotinamide riboside. Nicotinamide can be harmful to the liver in highdoses (as disclosed inhttp://www.livestrong.com/article/448906-therapeutic-levels-of-niacin-to-lower-cholesterol-levels/#ixzz2NO3KhDZu).The mass or molar amount of nicotinamide and/or nicotinamide metabolitescan be less than about 0.01, 0.1, 0.5, 1, 2, 5, or 10% of the totalcomposition. The mass or molar amount of nicotinamide and/ornicotinamide metabolites can be less than about 0.01, 0.1, 0.5, 1, 2, 5,or 10% of the total composition.

Without being limited to theory, ingestion of nicotinic acid and/ornicotinamide riboside and/or nicotinic acid metabolites can lower lipidcontent, lower triglyceride level, lower LDL level, lower totalcholesterol level, or increase HDL level. The ingestion of nicotinicacid and/or nicotinamide riboside and/or nicotinic acid metabolites canalso increase fat oxidation or stimulate sirtuin signaling, includingincrease activation of Sirt1 and Sirt3. In some embodiments, any of thecompositions described herein can include salts, derivatives,metabolites, catabolites, anabolites, precursors, and analogs ofnicotinic acid. For example, the metabolites can include nicotinyl CoA,nicotinuric acid, nicotinate mononucleotide, nicotinate adeninedinucleotide, or nicotinamide adenine dinucleotide. In some embodiments,the compositions cannot comprise nicotinamide. In some embodiments, thecompositions comprise nicotinamide. In some embodiments, thecompositions can be substantially free of nicotinic acid metabolites.

Leucine and Leucine Metabolites

The invention provides for compositions that include leucine and/orleucine metabolites. The leucine and/or leucine metabolites can be usedin free form. The term “free,” as used herein in reference to acomponent, indicates that the component is not incorporated into alarger molecular complex. For example a composition can include freeleucine that is not incorporated in a protein or freehydroxymethylbutyrate. The leucine can be L-leucine. The leucine and/orleucine metabolites can be in a salt form.

Without being limited to theory, ingestion of branched chain aminoacids, such as leucine, can stimulate sirtuin signaling, including Sirt1and Sirt3, as well as AMPK signaling, one or more of which can favorablymodulate inflammatory cytokine patterns. In some embodiments, any of thecompositions described herein can include salts, derivatives,metabolites, catabolites, anabolites, precursors, and analogs ofleucine. For example, the metabolites can include hydroxymethylbutyrate(HMB), keto-isocaproic acid (KIC), and keto isocaproate. The HMB can bein a variety of forms, including calcium 3-hydroxy-3-methylbutyratehydrate.

In certain embodiments of the invention, any of the compositionsdisclosed herein can be formulated such that they do not contain (orexclude) one or more amino acids selected from the group consisting oflysine, glutamate, proline, arginine, valine, isoleucine, aspartic acid,asparagine, glycine, threonine, serine, phenylalanine, tyrosine,histidine, alanine, tryptophan, methionine, glutamine, taurine,carnitine, cystine and cysteine.

In some embodiments, the compositions can be substantially free of oneor more, or all non-leucine amino acids. For example, the compositionscan be free of alanine, arginine, asparagine, aspartic acid, cysteine,glutamic acid, glutamine, glycine, histidine, isoleucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and/or valine.

The compositions can be substantially free of free non-leucine aminoacids such as alanine, arginine, asparagine, aspartic acid, cysteine,glutamic acid, glutamine, glycine, histidine, isoleucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and/or valine.

In some embodiments, the compositions can be substantially free of oneor more, or all of non-branched chain or non-leucine amino acids. Forexample, the compositions can be free of alanine, arginine, asparagine,aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, and/or tyrosine. In some embodiments, the compositions canbe substantially free of isoleucine and/or valine. The subjectcompositions can be substantially free of the individual amino acidsalanine, glycine, glutamic acid, and proline. The subject compositionscan be substantially free of one or more of the individual amino acidsalanine, glycine, glutamic acid, and proline. The subject compositionscan be substantially free of alanine. The subject compositions can besubstantially free of glycine. The subject compositions can besubstantially free of valine. The compositions can be substantially freeof any non-branched chain amino acids. The mass or molar amount of anon-branched chain amino acid can be less than about 0.01, 0.1, 0.5, 1,2, 5, or 10% of the total composition or of the total amino acids in thecomposition. The mass or molar amount of a non-leucine amino acid can beless than about 0.01, 0.1, 0.5, 1, 2, 5, or 10% of the total compositionor of the total amino acids in the composition.

For clarity, the amino acids described herein can be intact amino acidsexisting in free form or salt form thereof. For example, the subjectcompositions can be substantially free of free amino acids, such asalanine, glycine, glutamic acid, and proline. The mass or molar amountof a non-branched chain amino acid, any amino acid, or any non-leucineamino acid can be less than about 0.01, 0.1, 0.5, 1, 2, 5, or 10% of thetotal composition, of the total amino acids in the composition, or ofthe total free amino acids in the composition.

Therapeutic Agents

The subject compositions can further include one or morepharmaceutically active agents or therapeutic agents other thannicotinic acid and/or nicotinamide riboside and/or nicotinic acidmetabolites. The therapeutic agents or pharmaceutically active agentscan be any agent that is known in the art. For example, the combinationcompositions can further comprise a pharmaceutically activeanti-hyperlipidemic agent, or a dietary supplement that also effects onlipid content. The anti-hyperlipidemic agent can be an oral agent orinjectable agent. The anti-hyperlipidemic agents can be in asub-therapeutic amount in lowering levels of total lipid content ortriglyceride, LDL or cholesterol levels, or increasing the HDL level.The types of the anti-hyperlipidemic agents known in the art caninclude, but are not limited to, HMG-CoA inhibitors (or statins),fibrates, nicotinic acid, bile acid sequestrants (resins), cholesterolabsorption inhibitors (ezetimibe), lomitapide, phytosterols, orlistat orothers. The statin type anti-hyperlipidemic agents can include but arenot limited to: atorvastatin, fluvastatin, pravastatin, lovastatin,simvastatin, pitavastatin, cerivastatin, rosuvastatin, orlovastatin/niacin ER. The cholesterol absorption inhibitors can includebut are not limited to ezetimibe, and combination of ezetimibe withsimvastatin. The fibrate type of anti-hyperlipidemic agents can includebut are not limited to: gemfibrozil, fenofibrate, fenofibric acid,clofibrate, or micronized fenofibrate. The bile acid sequestrants caninclude but are not limited to: colestipol, cholestyramine, orcolesevelam. Other types of anti-hyperlipidemic agent can includedextrothyroxine sodium or icosapent. These examples are provided fordiscussion purposes only, and are intended to demonstrate the broadscope of applicability of the invention to a wide variety of drugs. Itis not meant to limit the scope of the invention in any way.

The subject composition can further comprise one or more therapeuticagents that are herbs and/or supplements. The herbs and/or supplementscan have therapeutic effects that are unproven scientifically. Theexamples of the herbs and/or the supplements can be, but are not limitedto: Acai, Alfalfa, Aloe, Aloe Vera, Aristolochic Acids, Asian Ginseng,Astragalus, Bacillus coagulans, Belladonna, Beta-carotene,Bifidobacteria, Bilberry, Bilberry, Biotin, Bitter Orange, Black Cohosh,Black Cohosh, Black psyllium, Black tea, Bladderwrack, Blessed thistle,Blond psyllium, Blueberry, Blue-green algae, Boron, Bromelain,Butterbur, Calcium, Calendula, Cancell/Cantron/Protocel, Cartilage(Bovine and Shark), Cassia cinnamon, Cat's Claw, Chamomile, Chasteberry,Chondroitin sulfate, Chromium, Cinnamon, Clove, Coenzyme Q-10, ColloidalSilver Products, Cranberry, Creatine, Dandelion, Dandelion, Devil'sclaw, DHEA, Dong quai, Echinacea, Ephedra, Essiac/Flor-Essence,Eucalyptus, European Elder (Elderberry), European Mistletoe, EveningPrimrose Oil, Fenugreek, Feverfew, Fish oil, Flaxseed, Flaxseed oil,Folate, Folic acid, Garlic, Ginger, Gingko, Ginseng, Glucosaminehydrochloride, Glucosamine sulfate, Goldenseal, Grape Seed Extract,Green Tea, Hawthorn, Hoodia, Horse Chestnut, Horsetail, HydrazineSulfate, Iodine, Iron, Kava, Lactobacillus, Laetrile/Amygdalin,L-arginine, Lavender, Licorice, Lycium, Lycopene, Magnesium, Manganese,Melatonin, Milk Thistle, Mistletoe Extracts, Noni, Oral Probiotics,Pantothenic acid (Vitamin B5), Passionflower, PC-SPES, Pennyroyal,Peppermint, Phosphate salts, Pomegranate, Propolis, Pycnogenol,Pyridoxine (Vitamin B6), Red Clover, Red yeast, Riboflavin (Vitamin B2),Roman chamomile, Saccharomyces boulardii, S-Adenosyl-L-Methionine(SAMe), Sage, Saw Palmetto, Selected Vegetables/Sun's Soup, Selenium,Senna, Soy, St. John's Wort, sweet orange essence, Tea Tree Oil,Thiamine (Vitamin B1), Thunder God Vine, Turmeric, Valerian, Vitamin A,Vitamin B12, Vitamin C, Vitamin D, Vitamin E, Vitamin K, Wild yam,Yohimbe, Zinc or 5-HTP.

The amount of pharmaceutical agent, or any other component used in acombination composition described herein, can be a used in an amountthat is sub-therapeutic. In some embodiments, using sub-therapeuticamounts of an agent or component can reduce the side-effects of theagent. Use of sub-therapeutic amounts can still be effective,particularly when used in synergy with other agents or components.

A sub-therapeutic amount of the agent or component can be such that itis an amount below which would be considered therapeutic. For example,FDA guidelines can suggest a specified level of dosing to treat aparticular condition, and a sub-therapeutic amount would be any levelthat is below the FDA suggested dosing level. The sub-therapeutic amountcan be about 1, 5, 10, 15, 20, 25, 30, 35, 50, 75, 90, or 95% less thanthe amount that is considered to be a therapeutic amount. Thetherapeutic amount can be assessed for individual subjects, or forgroups of subjects. The group of subjects can be all potential subjects,or subjects having a particular characteristic such as age, weight,race, gender, or physical activity level.

In the case of nicotinic acid administered alone to lower lipid content,the physician suggested starting dose is 1000-3000 mg daily, withsubject specific dosing having a range of 1 mg to a maximum of 1000 mgdaily when administered with leucine and/or leucine metabolites. Theparticular dosing for a subject can be determined by a clinician bytitrating the dose and measuring the therapeutic response. Thetherapeutic dosing level can be determined by measuring fasting plasmacholesterol and LDL levels without causing clinically significantcutaneous vasodilation. A sub-therapeutic amount can be any level thatwould be below the recommended dosing of nicotinic acid. For example, ifa subject's therapeutic dosing level is determined to be 700 mg daily, adose of 600 mg would be a sub-therapeutic amount. Alternatively, asub-therapeutic amount can be determined relative to a group of subjectsrather than an individual subject. For example, if the averagetherapeutic amount of nicotinic acid, nicotinamide riboside or nicotinicacid metabolites for subjects with weights over 300 lbs is 2000 mg, thena sub-therapeutic amount can be any amount below 2000 mg. In someembodiments, the dosing can be recommended by a healthcare providerincluding, but not limited to a patient's physician, nurse,nutritionist, pharmacist, or other health care professional. A healthcare professional can include a person or entity that is associated withthe health care system. Examples of health care professionals caninclude surgeons, dentists, audiologists, speech pathologists,physicians (including general practitioners and specialists), physicianassistants, nurses, midwives, pharmaconomists/pharmacists, dietitians,therapists, psychologists, physical therapists, phlebotomists,occupational therapists, optometrists, chiropractors, clinical officers,emergency medical technicians, paramedics, medical laboratorytechnicians, radiographers, medical prosthetic technicians socialworkers, and a wide variety of other human resources trained to providesome type of health care service.

In the case of nicotinic acid, nicotinamide riboside, or nicotinic acidmetabolites, the therapeutically effective level of the nicotinic acid,nicotinamide riboside, nicotinic acid metabolites can be a circulatinglevel between about 1-100 nM. A sub-therapeutic level of the nicotinicacid, nicotinamide riboside, or nicotinic acid metabolites, by itself orin any combination, can be any circulating level at least about, lessthan about, or more than about 1, 2.5, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 60, 70, 80, 90 or 100 nM. The sub-therapeutic level of thenicotinic acid and/or nicotinamide riboside and/or nicotinic acidmetabolites, in a subject composition formulated for administration canbe less than about 1, 5, 10, 20, 30, 50, 60, 70, 80, 90, 100, 125, 150,175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 750, 800, 900 or 1000mg of the nicotinic acid and/or nicotinamide riboside and/or nicotinicacid metabolites.

Any of the components described herein, including leucine, HMB, KIC,nicotinic acid, nicotinamide riboside, and resveratrol can be used in asubject composition in free form, isolated form, purified from a naturalsource, and/or purified or prepared from a synthetic source. The naturalsource can be an animal source or plant source. The components can bepure to at least about 95, 97, 99, 99.5, 99.9, 99.99, or 99.999%.

Dosing Amounts

The invention provides for compositions that are combinations ofisolated components, such as leucine, metabolites of leucine, such asHMB, nicotinic acid, nicotinamide riboside, and/or resveratrol, thathave been isolated from one or more sources. The invention provides forcompositions that are enriched in leucine, metabolites of leucine, suchas HMB, nicotinic acid and/or nicotinamide riboside, and/or resveratrol.The components can be isolated from natural sources or created fromsynthetic sources and then enriched to increase the purity of thecomponents. Additionally, leucine can be isolated from a natural sourceand then enriched by one or more separations. The isolated and enrichedcomponents, such as leucine, can then be combined and formulated foradministration to a subject.

In some embodiments, a composition comprises an amount of nicotinic acidand/or nicotinamide riboside and/or nicotinic acid metabolites. Theamount of nicotinic acid and/or nicotinamide riboside and/or nicotinicacid metabolites can be a subtherapeutic amount, and/or an amount thatis synergistic with one or more other compounds in the composition orone or more of the compounds administered simultaneously or in closetemporal proximity with the composition. In some embodiments, thenicotinic acid and/or nicotinamide riboside and/or nicotinic acidmetabolites is administered in a low dose, a medium dose, or a highdose, which describes the relationship between two doses, and generallydo not define any particular dose range. The compositions can beadministered to a subject such that the subject is administered aselected total daily dose of the composition. The total daily dose canbe determined by the sum of doses administered over a 24 hour period.

A dose, which can be a unit dose, can comprise about, more than about,or less than about 200, 250, 400, 500, 550, 600, 700, 800, 900, 1000,1100, 1250, 1300 or more mg of leucine. The leucine can be free leucine.In some embodiments, a unit dose can comprise at least about 1000 mg offree leucine. The composition can comprise between about 10-1250,200-1250, or 500-1250 mg of leucine. A dose, which can be a unit dose,can comprise about, more than about, or less than about 10, 15, 20, 25,30, 35, 40, 45, 50, 100, 200, 250, 400, 500, 550, 600, 700, 800, 900,1000, 1250, 1300 or more mg of a leucine metabolite, such as HMB or KIC.The leucine metabolite can be a free leucine metabolite. The compositioncan comprise between about 10-900, 50-750, or 400-650 mg of the leucinemetabolite, such as HMB or KIC. In some embodiments, a unit dose cancomprise at least about 400 mg of free HMB. The amount of leucine andleucine metabolites as described herein can be administered daily orsimultaneously. The amount as described herein can be administered inone dose or separately in multiple doses daily.

In some embodiments, a daily dose of leucine can be about, less thanabout, or more than about 0.25-3 or 0.5-3.0 g/day (e.g. 0.5, 0.75, 1,1.25, 1.5, 1.75, 2, 2.5, 3, or more g/day). A daily dose of HMB can beabout, less than about, or more than about 0.20-3.0 g/day (e.g. 0.2,0.4, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, or more g/day). A daily dose of KICcan be about, less than about, or more than about 0.2-3.0 g/day (e.g.0.2, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or more g/day).

The dose of leucine or metabolite thereof, can be a therapeutic dose.The dose of leucine or metabolite thereof can be a sub-therapeutic dose.A sub-therapeutic dose of leucine can be about, less than about, or morethan about 0.25-3.0 g (e.g. 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5,3, or more g). A sub-therapeutic dose of leucine can be about, less thanabout, or more than about 0.25-3.0 g/day (e.g. 0.25, 0.5, 0.75, 1, 1.25,1.5, 1.75, 2, 2.5, 3, or more g/day). In some embodiments, thecompositions comprises less than 3.0 g daily dosage of leucine. Asub-therapeutic dose of HMB can be about, less than about, or more thanabout 0.05-3.0 g (e.g. 0.05, 0.1, 0.2, 0.4, 0.5, 0.75, 1, 1.5, 2, 2.5,3, or more g). A sub-therapeutic dose of HMB can be about, less thanabout, or more than about 0.05-3.0 g/day (e.g. 0.05, 0.1, 0.2, 0.4, 0.5,0.75, 1, 1.5, 2, 2.5, 3, or more g/day). A sub-therapeutic dose of KICcan be about, less than about, or more than about 0.1-3.0 g (e.g. 0.1,0.2, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or more g). Asub-therapeutic dose of KIC can be about, less than about, or more thanabout 0.1-3.0 g/day (e.g. 0.1, 0.2, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75,2, 2.5, 3, or more g/day).

A dose, which can be a unit dose, can comprise nicotinic acid,nicotinamide riboside or nicotinic acid metabolites, that can be about,more than about, or less than about 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10,20, 40, 60, 80, 100, 200, 250, 400, 500, 800, 1000, or 1500 mg of thenicotinic acid, nicotinamide riboside, or nicotinic acid metabolites.The composition can comprise between about 1-100, 5-50, or 10-20 mg ofthe nicotinic acid and/or nicotinamide riboside and/or nicotinic acidmetabolites. In some embodiments, a unit dose can comprise at leastabout 1 mg of nicotinic acid and/or nicotinamide riboside and/ornicotinic acid metabolites. In some embodiments, a unit dose cancomprise less than 250 mg of nicotinic acid and/or nicotinamide ribosideand/or nicotinic acid metabolites. The dosage can be adjusted for theintended subject administered. For example, a dose that is suitable fora canine can be less than the dose that is suitable for a human. Theamount of nicotinic acid, nicotinamide riboside and/or nicotinic acidmetabolites as described herein can be administered daily orsimultaneously. The amount as described herein can be administered inone dose or separately in multiple doses daily.

In some embodiments, the composition comprises both nicotinic acid andnicotinamide riboside, and the total amount of nicotinic acid andnicotinamide riboside can be about, more than about, or less than about0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 40, 60, 80, 100, 200, 250, 400,500, 600, 800, 900, 1000, or 1500 mg.

In other embodiments, a daily dose of nicotinic acid and/or nicotinamideriboside and/or nicotinic acid metabolites can be about, more thanabout, or less than about 0.0001 mg/kg (mg of nicotinic acid and/ornicotinamide riboside and/or nicotinic acid metabolites/kg of thesubject receiving the dose), 0.005 mg/kg, 0.01 mg/kg, 0.5 mg/kg, 1mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 12.5 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, or more.

A dose, which can be a unit dose, can comprise about, less than about,or more than about 1, 5, 10, 25, 35, 50, 51, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, or more mg of resveratrol. The composition cancomprise between about 5-500, 30-250, or 35-100 mg of resveratrol. Insome embodiments, a unit dose can comprise at least about 35 mg ofresveratrol. The amount of resveratrol as described herein can beadministered daily or simultaneously. The amount as described herein canbe administered in one dose or separately in multiple doses daily.

A daily low dose of resveratrol can comprise about, less than about, ormore than about 0.5 mg/kg (mg of resveratrol/kg of the subject receivingthe dose), 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 12.5 mg/kg,15 mg/kg, 20 mg/kg, 25 mg/kg, 50 mg/kg, or more; a daily medium dose ofresveratrol can comprise about, less than about, or more than about 20mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, 200 mg/kg, 250 mg/kg, or more; and a daily high dose ofresveratrol can comprise about, less than about, or more than about 150mg/kg, 175 mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg,400 mg/kg, or more. The dosing range as defined to low, medium or highcan be dependent on the subject receiving the dose and vary from subjectto subject.

In some embodiments, a composition, which can be formulated as a unitdose, can comprise (a) at least about 250 mg of leucine and/or at leastabout 25 mg of the one or more leucine metabolites. The composition canfurther comprise at least about 35 mg of resveratrol.

In some embodiments of the invention, the combination compositions canhave a specified ratio of leucine and/or metabolites thereof tonicotinic acid and/or nicotinamide metabolites and/or nicotinic acidmetabolites. The specified ratio can provide for effective and/orsynergistic treatment of hyperlipidemic conditions, which, for example,can be measured as a reduction in total lipid content, reduction incholesterol level, reduction in triglyceride level, reduction in LDLlevel, reduction in body weight, and/or increase in HDL level. The ratioof leucine amino acids and/or metabolites thereof to a nicotinic acidand/or nicotinamide riboside and/or nicotinic acid metabolite can be amass ratio, a molar ratio, or a volume ratio.

In some embodiments, a composition can comprise (a) leucine and/ormetabolites thereof (including HMB) and (b) nicotinic acid and/ornicotinamide riboside and/or nicotinic acid metabolites, where the massratio of (a) to (b) can be about, less than about, or greater than about0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, or 800. In some embodiments, the massratio of (a) to (b) is at least about 25. In some embodiments, the massratio of (a) to (b) is at least about 50. The composition can alsocomprise a minimal amount of nicotinic acid and/or nicotinamide ribosideand/or nicotinic acid metabolites, such as 5, 10 or 50 mg of thenicotinic acid and/or nicotinamide riboside and/or nicotinic acidmetabolites or a range of nicotinic acid and/or nicotinamide ribosideand/or nicotinic acid metabolites amount, such as 5-250 mg of nicotinicacid and/or nicotinamide riboside and/or nicotinic acid metabolites.

In other embodiments, a composition can comprise (a) nicotinic acidand/or nicotinamide riboside and/or nicotinic acid metabolites and (b)resveratrol, where the mass ratio of (a) to (b) can be about, less thanabout, or greater than about 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 50,100, 200, 300, 350, 400, 450, 500, 550, 600, or 650.

In some embodiments, the dosing of leucine, any metabolites of leucine,nicotinic acid, nicotinamide riboside, any nicotinic acid metabolites,and resveratrol can be designed to achieve a specified physiologicalconcentration or circulating level of leucine, metabolites of leucine,nicotinic acid, nicotinamide riboside, metabolites of nicotinic acidand/or resveratrol. The physiological concentration can be a circulatinglevel as measured in the serum or blood stream of a subject. The subjectcan be a human or an animal. A selected dosing can be altered based onthe characteristics of the subject, such as weight, rate of energymetabolism, genetics, ethnicity, height, or any other characteristic.

In some embodiments, a selected dose of a composition can beadministered to a subject such that the subject achieves a desiredcirculating level of the composition. The desired circulating level of acomponent can be either a therapeutically effective level or asub-therapeutic level.

The amount of leucine in a unit dose can be such that the circulatinglevel of leucine in a subject is about or greater than about 0.25 mM,0.5 mM, 0.75 mM, or 1 mM. A dosing of about 1,125 mg leucine (e.g., freeleucine), can achieve a circulating level of leucine in a subject thatis about 0.5 mM. A dosing of about 300 mg leucine (e.g., free leucine),can achieve a circulating level of leucine in a subject that is about0.25 mM.

The desired circulating level of the composition can be at least about0.25, 0.5, 0.75, 1 mM or more of leucine. The desired circulating levelof the composition can be at least about, less than about, or more thanabout 0.1, 0.25, 0.5, 0.75, 1, 10, 20, 40, 60 μM or more of a leucinemetabolite (such as HMB). The desired circulating level of thecomposition can be at least about 0.25, 0.5, 0.75, 1 mM or more of KIC.

The desired circulating level of the composition can be at least about,less than about, or more than about 0.1, 0.25, 0.5, 0.75, 1, 10, 20, 40,60, 80, 100, 120, 200, 400, 500, 1000, 1500, 2000, 2500, or 3000 nM ormore of the nicotinic acid and/or nicotinamide riboside and/or nicotinicacid metabolites. The therapeutically effective level of nicotinic acidand/or nicotinamide riboside and/or nicotinic acid metabolites can bebetween 44-111 μM, which corresponds to about 10-20 μg/mL.

The desired circulating level of the composition can be at least about,less than about, or more than about 40, 60, 80, 100, 120, 150, 200, 300,400, 800, 1600, 3000, or 5000 nM or more of the resveratrol. Theselected dose can be chosen based on the characteristics of the subject,such as weight, height, ethnicity, or genetics.

In some embodiments, a composition comprises leucine and nicotinic acidin amounts that are effective to achieve a circulating level of about0.3-1 mM leucine and about 1-100 nM nicotinic acid in a subject.

An oral dosing of about 1,125 mg leucine can achieve a circulating levelof leucine in a subject that is about 0.5 mM leucine. An oral dosing ofabout 300 mg leucine can achieve a circulating level of leucine in asubject that is about 0.25 mM.

An oral dosing of about 500 mg of HMB can achieve a circulating level ofHMB in a subject that is about 5 μM HMB. An oral dosing of about 100 mgof HMB can achieve a circulating level of HMB in a subject that is about0.8 μM HMB.

An oral dosing of about 3,000 mg nicotinic acid or nicotinamide ribosidecan achieve a circulating level of nicotinic acid or nicotinamideriboside in a subject that is about 10 μM nicotinic acid or nicotinamideriboside. An oral dosing of about 50 mg nicotinic acid or nicotinamideriboside can achieve a circulating level of nicotinic acid ornicotinamide riboside in a subject that is about 10-100 nM nicotinicacid or nicotinamide riboside.

An oral dosing of about 1100 mg of resveratrol can achieve a circulatinglevel of resveratrol in a subject that is about 0.5 mM resveratrol. Anoral dosing of about 50 mg of resveratrol can achieve a circulatinglevel of resveratrol in a subject that is about 200 nM resveratrol.

In some embodiments, the compositions can be formulated to achieve adesired circulating molar or mass ratios achieved after administrationone or more compositions to a subject. The compositions can be acombination composition described herein. The molar ratio can beadjusted to account for the bioavailability, the uptake, and themetabolic processing of the one or more components of a combinationcomposition. For example, if the bioavailability of a component is low,then the molar amount of a that component can be increased relative toother components in the combination composition. In some embodiments,the circulating molar or mass ratio is achieved within about 0.1, 0.5,0.75, 1, 3, 5, or 10, 12, 24, or 48 hours after administration. Thecirculating molar or mass ratio can be maintained for a time period ofabout or greater than about 0.1, 1, 2, 5, 10, 12, 18, 24, 36, 48, 72, or96 hours.

In some embodiments, the circulating molar ratio of leucine to nicotinicacid or nicotinamide riboside is about, less than about, or greater thanabout 1, 5, 10, 20, 50, 100, 500, 1000, 5000, or 10000. In someembodiments, the circulating molar ratio of HMB to nicotinic acid ornicotinamide riboside is about or greater than about, or less than about0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 50, or 100. In some embodiments, thecirculating molar ratio of a nicotinic acid or nicotinamide riboside toresveratrol is about, less than about, or greater than about 0.01, 0.05,0.1, 0.5, 1, 5, 10, 20, 50, or 100.

Dosing Forms

The compositions described herein can be compounded into a variety ofdifferent dosage forms. It can be used orally as a tablet, a capsule, apill, a granule, an emulsion, a gel, a plurality of beads encapsulatedin a capsule, a powder, a suspension, a liquid, a semi-liquid, asemi-solid, a syrup, a slurry, a chewable form, caplets, soft gelatincapsules, lozenges or solution. Alternatively, the compositions can beformulated for inhalation or for intravenous delivery. The compositionscan also be formulated as a nasal spray or for injection when insolution form. In some embodiments, the composition can be a liquidcomposition suitable for oral consumption.

Compositions formulated for inhalation can be packaged in an inhalerusing techniques known in the art. An inhaler can be designed todispense 0.25, 0.5, or 1 unit dose per inhalation. An inhaler can have acanister that holds the subject composition formulated for inhalation, ametering valve that allows for a metered quantity of the formulation tobe dispensed with each actuation, and an actuator or mouthpiece thatallows for the device to be operated and direct the subject compositioninto the subject's lungs. The formulated composition can include aliquefied gas propellant and possibly stabilizing excipients. Theactuator can have a mating discharge nozzle that connects to thecanister and a dust cap to prevent contamination of the actuator. Uponactuation, the subject composition can be volatized, which results inthe formation of droplets of the subject composition. The droplets canrapidly evaporate resulting in micrometer-sized particles that are theninhaled by the subject Inhalers and methods for formulating compositionsfor inhalation are described in are described in U.S. Pat. Nos.5,069,204, 7,870,856 and U.S. Patent Application No. 2010/0324002, whichare incorporated herein by reference in its entirety.

Compositions of the invention suitable for oral administration can bepresented as discrete dosage forms, such as capsules, cachets, ortablets, or liquids or aerosol sprays each containing a predeterminedamount of an active ingredient as a powder or in granules, a solution,or a suspension in an aqueous or non-aqueous liquid, an oil-in-wateremulsion, or a water-in-oil liquid emulsion, including liquid dosageforms (e.g., a suspension or slurry), and oral solid dosage forms (e.g.,a tablet or bulk powder). Oral dosage forms can be formulated astablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilicsuspensions, liquids, gels, syrups, slurries, suspensions and the like,for oral ingestion by an individual or a patient to be treated. Suchdosage forms can be prepared by any of the methods of formulation. Forexample, the active ingredients can be brought into association with acarrier, which constitutes one or more necessary ingredients. Capsulessuitable for oral administration include push-fit capsules made ofgelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredients in admixture with filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. Optionally, the inventivecomposition for oral use can be obtained by mixing a composition a solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). In general,the compositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product into the desiredpresentation. For example, a tablet can be prepared by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets can be prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as powder or granules, optionallymixed with an excipient such as, but not limited to, a binder, alubricant, an inert diluent, and/or a surface active or dispersingagent. Molded tablets can be made by molding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.

The liquid forms, in which the formulations disclosed herein can beincorporated for administration orally or by injection, include aqueoussolution, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, or peanut oil as well as elixirs and similar pharmaceuticalvehicles. Suitable dispersing or suspending agents for aqueoussuspensions include synthetic natural gums, such as tragacanth, acacia,alginate, dextran, sodium carboxymethyl cellulose, methylcellulose,polyvinylpyrrolidone or gelatin.

A subject can be treated by combination of an injectable composition andan orally ingested composition.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for reconstitution with water or other suitable vehiclesbefore use. Such liquid preparations can be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, methyl cellulose or hydrogenated ediblefats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters or ethyl alcohol); preservatives(e.g., methyl or propyl p-hydroxybenzoates or sorbic acid); andartificial or natural colors and/or sweeteners.

The preparation of pharmaceutical compositions of this invention,including oral and inhaled formulations, can be conducted in accordancewith generally accepted procedures for the preparation of pharmaceuticalpreparations. See, for example, Remington's Pharmaceutical Sciences 18thEdition (1990), E. W. Martin ed., Mack Publishing Co., PA. Depending onthe intended use and mode of administration, it can be desirable toprocess the magnesium-counter ion compound further in the preparation ofpharmaceutical compositions. Appropriate processing can include mixingwith appropriate non-toxic and non-interfering components, sterilizing,dividing into dose units, and enclosing in a delivery device.

This invention further encompasses anhydrous compositions and dosageforms comprising an active ingredient, since water can facilitate thedegradation of some compounds. For example, water can be added (e.g.,5%) in the arts as a means of simulating long-term storage in order todetermine characteristics such as shelf-life or the stability offormulations over time. Anhydrous compositions and dosage forms of theinvention can be prepared using anhydrous or low moisture containingingredients and low moisture or low humidity conditions. Compositionsand dosage forms of the invention which contain lactose can be madeanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected. An anhydrouscomposition can be prepared and stored such that its anhydrous nature ismaintained. Accordingly, anhydrous compositions can be packaged usingmaterials known to prevent exposure to water such that they can beincluded in suitable formulary kits. Examples of suitable packaginginclude, but are not limited to, hermetically sealed foils, plastic orthe like, unit dose containers, blister packs, and strip packs.

An ingredient described herein can be combined in an intimate admixturewith a pharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier can take a wide variety of formsdepending on the form of preparation desired for administration. Inpreparing the compositions for an oral dosage form, any of the usualpharmaceutical media can be employed as carriers, such as, for example,water, glycols, oils, alcohols, flavoring agents, preservatives,coloring agents, and the like in the case of oral liquid preparations(such as suspensions, solutions, and elixirs) or aerosols; or carrierssuch as starches, sugars, micro-crystalline cellulose, diluents,granulating agents, lubricants, binders, and disintegrating agents canbe used in the case of oral solid preparations, in some embodimentswithout employing the use of lactose. For example, suitable carriersinclude powders, capsules, and tablets, with the solid oralpreparations. If desired, tablets can be coated by standard aqueous ornonaqueous techniques.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

Binders suitable for use in dosage forms include, but are not limitedto, corn starch, potato starch, or other starches, gelatin, natural andsynthetic gums such as acacia, sodium alginate, alginic acid, otheralginates, powdered tragacanth, guar gum, cellulose and its derivatives(e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulosecalcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methylcellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose,microcrystalline cellulose, and mixtures thereof.

Lubricants which can be used to form compositions and dosage forms ofthe invention include, but are not limited to, calcium stearate,magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol,mannitol, polyethylene glycol, other glycols, stearic acid, sodiumlauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil,cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, ormixtures thereof. Additional lubricants include, for example, a syloidsilica gel, a coagulated aerosol of synthetic silica, or mixturesthereof. A lubricant can optionally be added, in an amount of less thanabout 1 weight percent of the composition.

Lubricants can be also be used in conjunction with tissue barriers whichinclude, but are not limited to, polysaccharides, polyglycans,seprafilm, interceed and hyaluronic acid.

Disintegrants can be used in the compositions of the invention toprovide tablets that disintegrate when exposed to an aqueousenvironment. Too much of a disintegrant can produce tablets which candisintegrate in the bottle. Too little can be insufficient fordisintegration to occur and can thus alter the rate and extent ofrelease of the active ingredient(s) from the dosage form. Thus, asufficient amount of disintegrant that is neither too little nor toomuch to detrimentally alter the release of the active ingredient(s) canbe used to form the dosage forms of the compounds disclosed herein. Theamount of disintegrant used can vary based upon the type of formulationand mode of administration, and can be readily discernible to those ofordinary skill in the art. About 0.5 to about 15 weight percent ofdisintegrant, or about 1 to about 5 weight percent of disintegrant, canbe used in the pharmaceutical composition. Disintegrants that can beused to form compositions and dosage forms of the invention include, butare not limited to, agar-agar, alginic acid, calcium carbonate,microcrystalline cellulose, croscarmellose sodium, crospovidone,polacrilin potassium, sodium starch glycolate, potato or tapioca starch,other starches, pre-gelatinized starch, other starches, clays, otheralgins, other celluloses, gums or mixtures thereof.

Examples of suitable fillers for use in the compositions and dosageforms disclosed herein include, but are not limited to, talc, calciumcarbonate (e.g., granules or powder), microcrystalline cellulose,powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol,starch, pre-gelatinized starch, and mixtures thereof.

When aqueous suspensions and/or elixirs are desired for oraladministration, the active ingredient therein can be combined withvarious sweetening or flavoring agents, coloring matter or dyes and, ifso desired, emulsifying and/or suspending agents, together with suchdiluents as water, ethanol, propylene glycol, glycerin and variouscombinations thereof.

The tablets can be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate canbe employed. Formulations for oral use can also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example, peanut oil, liquidparaffin or olive oil.

In one embodiment, the composition can include a solubilizer to ensuregood solubilization and/or dissolution of the compound of the presentinvention and to minimize precipitation of the compound of the presentinvention. This can be especially important for compositions fornon-oral use, e.g., compositions for injection. A solubilizer can alsobe added to increase the solubility of the hydrophilic drug and/or othercomponents, such as surfactants, or to maintain the composition as astable or homogeneous solution or dispersion.

The composition can further include one or more pharmaceuticallyacceptable additives and excipients. Such additives and excipientsinclude, without limitation, detackifiers, anti-foaming agents,buffering agents, polymers, antioxidants, preservatives, chelatingagents, viscomodulators, tonicifiers, flavorants, colorants, odorants,opacifiers, suspending agents, binders, fillers, plasticizers,lubricants, and mixtures thereof. A non-exhaustive list of examples ofexcipients includes monoglycerides, magnesium stearate, modified foodstarch, gelatin, microcrystalline cellulose, glycerin, stearic acid,silica, yellow beeswax, lecithin, hydroxypropylcellulose, croscarmellosesodium, and crospovidone.

The compositions described herein can also be formulated asextended-release, sustained-release or time-release such that one ormore components are released over time. Delayed release can be achievedby formulating the one or more components in a matrix of a variety ofmaterials or by microencapsulation. The compositions can be formulatedto release one or more components over a time period of 1, 4, 6, 8, 12,16, 20, 24, 36, or 48 hours. The release of the one or more componentscan be at a constant or changing rate.

In some embodiments, a subject composition described herein can beformulated in as matrix pellets in which particles of the subjectcomposition are embedded in a matrix of water-insoluble plastic andwhich are enclosed by a membrane of water-insoluble plastic containingembedded particles of lactose, produces and maintains plasma levels ofthe subject composition within the targeted therapeutic range. In otherembodiments, a subject composition can be formulated as a sustainedrelease tablet obtained by coating core granules composed mainly of thesubject composition with a layer of a coating film composed of ahydrophobic material and a plastic excipient and optionally containingan enteric polymer material to form coated granules and then bycompressing the coated granules together with a disintegratingexcipient. Sustained release formulations are described in U.S. Pat.Nos. 4,803,080, and 6,426,091, which are herein incorporated byreference in its entirety.

Using the controlled release dosage forms provided herein, the one ormore cofactors can be released in its dosage form at a slower rate thanobserved for an immediate release formulation of the same quantity ofcomponents. In some embodiments, the rate of change in the biologicalsample measured as the change in concentration over a defined timeperiod from administration to maximum concentration for an controlledrelease formulation is less than about 80%, 70%, 60%, 50%, 40%, 30%,20%, or 10% of the rate of the immediate release formulation.Furthermore, in some embodiments, the rate of change in concentrationover time is less than about 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%of the rate for the immediate release formulation.

In some embodiments, the rate of change of concentration over time isreduced by increasing the time to maximum concentration in a relativelyproportional manner. For example, a two-fold increase in the time tomaximum concentration can reduce the rate of change in concentration byapproximately a factor of 2. As a result, the one or more cofactors canbe provided so that it reaches its maximum concentration at a rate thatis significantly reduced over an immediate release dosage form. Thecompositions of the present invention can be formulated to provide ashift in maximum concentration by 24 hours, 16 hours, 8 hours, 4 hours,2 hours, or at least 1 hour. The associated reduction in rate of changein concentration can be by a factor of about 0.05, 0.10, 0.25, 0.5 or atleast 0.8. In certain embodiments, this is accomplished by releasingless than about 30%, 50%, 75%, 90%, or 95% of the one or more cofactorsinto the circulation within one hour of such administration.

Optionally, the controlled release formulations exhibit plasmaconcentration curves having initial (e.g., from 2 hours afteradministration to 4 hours after administration) slopes less than 75%,50%, 40%, 30%, 20% or 10% of those for an immediate release formulationof the same dosage of the same cofactor.

In some embodiments, the rate of release of the cofactor as measured indissolution studies is less than about 80%, 70%, 60% 50%, 40%, 30%, 20%,or 10% of the rate for an immediate release formulation of the samecofactor over the first 1, 2, 4, 6, 8, 10, or 12 hours.

The controlled release formulations provided herein can adopt a varietyof formats. In some embodiments, the formulation is in an oral dosageform, including liquid dosage forms (e.g., a suspension or slurry), andoral solid dosage forms (e.g., a tablet or bulk powder), such as, butnot limited to those, those described herein.

The controlled release tablet of a formulation disclosed herein can beof a matrix, reservoir or osmotic system. Although any of the threesystems is suitable, the latter two systems can have more optimalcapacity for encapsulating a relatively large mass, such as for theinclusion of a large amount of a single cofactor, or for inclusion of aplurality of cofactors, depending on the genetic makeup of theindividual. In some embodiments, the slow-release tablet is based on areservoir system, wherein the core containing the one or more cofactorsis encapsulated by a porous membrane coating which, upon hydration,permits the one or more cofactors to diffuse through. Because thecombined mass of the effective ingredients is generally in gramquantity, an efficient delivery system can provide optimal results.

Thus, tablets or pills can also be coated or otherwise compounded toprovide a dosage form affording the advantage of prolonged action. Forexample, the tablet or pill can comprise an inner dosage an outer dosagecomponent, the latter being in the form of an envelope over the former.The two components can be separated by an enteric layer which serves toresist disintegration in the stomach and permits the inner component topass intact into the duodenum or to be delayed in release. A variety ofmaterials can be used for such enteric layers or coatings such materialsincluding a number of polymeric acids and mixtures of polymeric acidswith such materials as shellac, cetyl alcohol and cellulose acetate. Insome embodiments, a formulation comprising a plurality of cofactors canhave different cofactors released at different rates or at differenttimes. For example, there can be additional layers of cofactorsinterspersed with enteric layers.

Methods of making sustained release tablets are known in the art, e.g.,see U.S. Patent Publications 2006/051416 and 2007/0065512, or otherreferences disclosed herein. Methods such as described in U.S. Pat. Nos.4,606,909, 4,769,027, 4,897,268, and 5,395,626 can be used to preparesustained release formulations of the one or more cofactors determinedby the genetic makeup of an individual. In some embodiments, theformulation is prepared using OROS® technology, such as described inU.S. Pat. Nos. 6,919,373, 6,923,800, 6,929,803, and 6,939,556. Othermethods, such as described in U.S. Pat. Nos. 6,797,283, 6,764,697, and6,635,268, can also be used to prepare the formulations disclosedherein.

In some embodiments, the compositions can be formulated in a foodcomposition. For example, the compositions can be a beverage or otherliquids, solid food, semi-solid food, with or without a food carrier.For example, the compositions can include a black tea supplemented withany of the compositions described herein. The composition can be a dairyproduct supplemented any of the compositions described herein. In someembodiments, the compositions can be formulated in a food composition.For example, the compositions can comprise a beverage, solid food,semi-solid food, or a food carrier.

In some embodiments, liquid food carriers, such as in the form ofbeverages, such as supplemented juices, coffees, teas, sodas, flavoredwaters, and the like can be used. For example, the beverage can comprisethe formulation as well as a liquid component, such as various deodorantor natural carbohydrates present in conventional beverages. Examples ofnatural carbohydrates include, but are not limited to, monosaccharidessuch as, glucose and fructose; disaccharides such as maltose andsucrose; conventional sugars, such as dextrin and cyclodextrin; andsugar alcohols, such as xylitol and erythritol. Natural deodorant suchas taumatin, stevia extract, levaudioside A, glycyrrhizin, and syntheticdeodorant such as saccharin and aspartame can also be used. Agents suchas flavoring agents, coloring agents, and others can also be used. Forexample, pectic acid and the salt thereof, alginic acid and the saltthereof, organic acid, protective colloidal adhesive, pH controllingagent, stabilizer, a preservative, glycerin, alcohol, or carbonizingagents can also be used. Fruit and vegetables can also be used inpreparing foods or beverages comprising the formulations discussedherein.

Alternatively, the compositions can be a snack bar supplemented with anyof the compositions described herein. For example, the snack bar can bea chocolate bar, a granola bar, or a trail mix bar. In yet anotherembodiment, the present dietary supplement or food compositions areformulated to have suitable and desirable taste, texture, and viscosityfor consumption. Any suitable food carrier can be used in the presentfood compositions. Food carriers of the present invention includepractically any food product. Examples of such food carriers include,but are not limited to food bars (granola bars, protein bars, candybars, etc.), cereal products (oatmeal, breakfast cereals, granola,etc.), bakery products (bread, donuts, crackers, bagels, pastries,cakes, etc.), beverages (milk-based beverage, sports drinks, fruitjuices, alcoholic beverages, bottled waters), pastas, grains (rice,corn, oats, rye, wheat, flour, etc.), egg products, snacks (candy,chips, gum, chocolate, etc.), meats, fruits, and vegetables. In anembodiment, food carriers employed herein can mask the undesirable taste(e.g., bitterness). Where desired, the food composition presented hereinexhibit more desirable textures and aromas than that of any of thecomponents described herein. For example, liquid food carriers can beused according to the invention to obtain the present food compositionsin the form of beverages, such as supplemented juices, coffees, teas,and the like. In other embodiments, solid food carriers can be usedaccording to the invention to obtain the present food compositions inthe form of meal replacements, such as supplemented snack bars, pasta,breads, and the like. In yet other embodiments, semi-solid food carrierscan be used according to the invention to obtain the present foodcompositions in the form of gums, chewy candies or snacks, and the like.

The dosing of the combination compositions can be administered about,less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore times a daily. A subject can receive dosing for a period of about,less than about, or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 or more days, weeks or months. A unit dose can be afraction of the daily dose, such as the daily dose divided by the numberof unit doses to be administered per day. A unit dose can be a fractionof the daily dose that is the daily dose divided by the number of unitdoses to be administered per day and further divided by the number ofunit doses (e.g. tablets) per administration. The number of unit dosesper administration can be about, less than about, or more than about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The number of doses per day can beabout, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more. The number of unit doses per day can be determined bydividing the daily dose by the unit dose, and can be about, less thanabout, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 6, 17, 18, 19, 20, or more unit doses per day. For example, a unitdose can be about ½, ⅓, ¼, ⅕, ⅙, 1/7, ⅛, 1/9, 1/10. A unit dose can beabout one-third of the daily amount and administered to the subjectthree times daily. A unit dose can be about one-half of the daily amountand administered to the subject twice daily. A unit dose can be aboutone-fourth of the daily amount with two unit doses administered to thesubject twice daily. In some embodiments, a unit dose comprises about,less than about, or more than about 50 mg resveratrol. In someembodiments, a unit dose comprises about, less than about, or more thanabout 550 mg leucine. In some embodiments, a unit dose comprises about,less than about, or more than about 200 mg of one or more leucinemetabolites.

In some embodiments, a unit dose (e.g. a unit dose comprising one ormore leucine metabolites, such as HMB) is administered as one unit dosetwo times per day. A unit dose can comprise more than one capsule,tablet, vial, or entity.

Compositions disclosed herein can further comprise a flavorant and canbe a solid, liquid, gel or emulsion.

When the subject composition administered further comprises one or moretherapeutic agents, and the therapeutic agents have a shorter half-lifethan the leucine and/or leucine metabolites, or the nicotinic acidand/or nicotinamide riboside and/or nicotinic acid metabolites, the unitdose forms of the therapeutic agent and the leucine and/or leucinemetabolites, or nicotinic acid and/or nicotinamide riboside and/ornicotinic acid metabolites can be adjusted accordingly.

Methods

The subject composition is particularly useful for ameliorating ahyperlipidemic condition. In one embodiment, the invention provides formethods for reducing total lipid content or lowering level of totalcholesterol, LDL, or triglyceride, increasing HDL level, or reducingatherosclerotic plaque size comprising administering to a subject inneed thereof any of the subject compositions. The level or contentdescribed herein can be a circulating concentration in serum or bloodstream, or a total amount in the subject's body. In some embodiments,the subject composition is useful in increasing weight loss of thesubject, and increase sirt1 activation or fat oxidation of the subject.In various embodiments of the invention, a composition is administeredto the subject in an amount that delivers synergizing amounts of leucineand/or a metabolite thereof, nicotinic acid and/or nicotinamide ribosideand/or a nicotinic acid metabolite, and/or resveratrol sufficient toameliorate a hyperlipidemic condition of the subject. In someembodiments, nicotinic acid, nicotinamide riboside or nicotinic acidmetabolites can induce a side effect (e.g., cutaneous vasodilation) ifit is administered to a subject at its therapeutic dose without leucineor leucine metabolites. Methods described herein can also be useful forameliorating the side-effect without losing the therapeuticeffectiveness of nicotinic acid, nicotinamide riboside or nicotinic acidmetabolites. A description of various aspects, features, embodiments,and examples, is provided herein.

The subject methods comprising the use of leucine and/or leucinemetabolite with nicotinic acid and/or nicotinamide riboside and/ornicotinic acid metabolites can be applicable for administering to asubject that is suffering from hyperlipidemia, at risk of suffering fromhyperlipidemia, and/or suffering from a condition that is associatedwith hyperlipidemia such as cardiovascular conditions. In some cases, aneffective amount of an additional therapeutic or a pharmaceuticallyactive agent that is known in the medical art (e.g., ananti-hyperlipidemic agent) can be administered to a subject inconjunction with any of the subject compositions.

Hyperlipidemia can be characterized by a high level of total lipidcontent or level in a subject. Hyperlipidemia can also be characterizedby a high level of body weight or BMI of a subject. The types of lipidcan include cholesterol, cholesterol esters, phospholipids andtriglycerides. The content or level of the lipids can be a circulatinglevel that is measured in the bloodstream, plasma or serum of thesubject. The content of the lipids can also be correlated by the bodyweight of the subject. These lipids can be transported in the blood aslarge lipoproteins including chylomicrons, very low-density lipoproteins(VLDL), intermediate-density lipoprotein (IDL), low-density lipoproteins(LDL) and high-density lipoproteins (HDL) based on their density. Mosttriglycerides can be transported in chylomicrons or VLDL and mostcholesterol can be carried in LDL and HDL. High levels of lipid in thecirculation can cause lipid accumulation on the walls of arteries, andfurther result in atherosclerotic plaque formation and therefore narrowthe arteries. The subject that is suffering from hyperlipidemia can beat high risk of acquiring a cardiovascular condition. Hyperlipidemia canalso be characterized by a high level of some lipoproteins or a lowlevel of HDL. The condition that the subject is suffering from or atrisk of suffering from can be a condition that is associated with anabnormal level of lipoproteins or lipids in the subject. The subjectcomposition can be used to change the level of the one or more lipids orlipoproteins in the subject. In some embodiments, the type of lipids orlipoproteins that its level can be affected by the subject compositionsand methods can be one or more lipoproteins and/or lipids including butnot limited to: total cholesterol, triglyceride, HDL, IDL, VLDL or LDL.

A number of methods can be used to assess the levels of lipoproteinsand/or lipids in a subject. These methods can differ from one another inthe type of sample and the analytical technique used. The type of samplethat can be used to measure such levels include but are not limited to:serum, plasma, whole blood, red blood cells or tissue samples. Wheredesired, the level of lipoproteins and/or lipids can be measured under afasting condition, e.g., without taking food for at least about 8 hours,10 hours, 12 hours, 15 hours, 24 hours, or even longer.

The size of atherosclerotic plaque or lesion can be measured by anymethods that are known in the art. For examples, methods described inPhan B A et al., “Effects of niacin on glucose levels, coronary stenosisprogression, and clinical events in subjects with normal baselineglucose levels (100 mg/dl): a combined analysis of the FamilialAtherosclerosis Treatment Study (FATS), HDL-Atherosclerosis TreatmentStudy (HATS), Armed Forces Regression Study (AFREGS), and Carotid PlaqueComposition by MRI during lipid-lowering (CPC) study”, Am J Cardiol.2013 Feb. 1; 111(3):352-5, and Lehman S J et al., “Assessment ofCoronary Plaque Progression in Coronary CT Angiography Using aSemi-Quantitative Score”, JACC Cardiovasc Imaging. 2009 November; 2(11):1262-1270. Non-limiting example of the method to measure the size ofatherosclerotic plaque or lesion can be quantitative coronaryangiography.

In some embodiments, the amounts of the nicotinic acid, nicotinamideriboside and/or nicotinic acid metabolites in the composition, ifadministered to a subject alone and without leucine, a leucinemetabolite, or resveratrol, can cause no therapeutic effect in thesubject. Additionally, the amounts of leucine, a leucine metabolite, orresveratrol, if administered to the subject without the nicotinic acid,nicotinamide riboside or nicotinic acid metabolites, can have notherapeutic effect on the subject. However, when the nicotinic acid,nicotinamide riboside and/or nicotinic acid metabolites is administeredin conjunction with either leucine, a leucine metabolite, orresveratrol, a therapeutic effect can be observed. The “therapeuticeffect” described herein is a lowered total lipid content, decreasedtotal cholesterol level, decreased triglyceride level, increased HDLlevel, decreased LDL level or reduced atherosclerotic plaque in thesubject administered. Accordingly, the invention provides a method foradministering a composition comprising (a) leucine and/or one or moremetabolites thereof and (b) nicotinic acid and/or nicotinamide ribosideand/or nicotinic acid metabolites present in a sub-therapeutic amount,wherein the composition is effective in increasing treatinghyperlipidemic conditions as compared to that of component (b) when itis used alone. The amount of leucine in the composition can also be asub-therapeutic amount.

Quantification of the therapeutic effect can show that the effect of acomposition that comprises (a) leucine or a leucine metabolite and (b) asub-therapeutic amount of nicotinic acid, nicotinamide riboside or anicotinic acid metabolite is greater than the predicted effect ofadministering (a) or (b) alone, assuming simple additive effects of (a)and (b), and thus the effect is synergistic. The synergistic effect canbe quantified as the measured effect above the predicted simple additiveeffect of the components of the composition. For example, ifadministration of component (a) alone yields an effect of 10% relativeto control, administration of component (b) alone yields an effect of15% relative to control, and administration of a composition comprisingboth (a) and (b) yields an effect of 60% relative to control, thesynergistic effect would be 60%−(15%+10%), or 35%.

In some embodiments, a therapeutic amount of nicotinic acid,nicotinamide riboside and/or nicotinic acid metabolites can cause a sideeffect that can be characterized by an increased in cutaneousvasodilation. The increase in the cutaneous vasodilation can beclinically significant. A sub-therapeutic amount of nicotinic acid,nicotinamide riboside and/or nicotinic acid metabolites cannot cause aclinically significant cutaneous vasodilation, or can reduce the degreeof cutaneous vasodilation in the subject administered as compared to atherapeutic amount of nicotinic acid, nicotinamide riboside and/ornicotinic acid metabolites. The subject compositions and methodsdescribed herein comprise a sub-therapeutic amount of nicotinic acid,nicotinamide riboside and/or nicotinic acid metabolites, to be used withleucine and/or leucine metabolites to result a therapeutic degree ofeffect of the sub-therapeutic amount of the nicotinic acid, nicotinamideriboside and/or nicotinic acid metabolites without causing the degree ofside effect that can normally be caused by a therapeutic amount ofnicotinic acid, nicotinamide riboside and/or nicotinic acid metaboliteswhen used without leucine and/or leucine metabolites. Levels ofcutaneous vasodilation can be measured by any methods known in themedical art, such as the methods including laser-Doppler flowmeter. Withthe same level of therapeutic effect (e.g. lowering cholesterol level byat least 5%), the level of cutaneous vasodilation caused by the subjectcompositions as compared to nicotinic acid, nicotinamide riboside and/ornicotinic acid metabolites without leucine and/or leucine metabolitescan be lower. For example, less than about 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90% of the level that iscaused by a therapeutic amount of to nicotinic acid, nicotinamideriboside and/or nicotinic acid metabolites.

The amount of leucine and/or leucine metabolites can be at least about25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500 mg. Thesub-therapeutic amount of nicotinic acid, nicotinamide riboside, and/ornicotinic acid metabolites can be less than 1 g, 500, 250, 100, 50 or 10mg. The amount of nicotinic acid, nicotinamide riboside, and/ornicotinic acid metabolites can be between about 1-100 mg. The amount ofnicotinic acid, nicotinamide riboside, and/or nicotinic acid metabolitescan be capable of achieving a circulating level of nicotinic acid,nicotinamide riboside, and/or nicotinic acid metabolites that is about1-100 nM, higher than about 100 nM or at least about 10 nM.

Accordingly, the multi-component compositions described herein (such asnicotinic acid/leucine, nicotinic acid/leucine/resveratrol, nicotinamideriboside/leucine, and nicotinamide riboside/leucine/resveratrol) canhave a beneficial or synergistic effect on lowering total lipid content,decreasing total cholesterol level, decreasing triglyceride level,increasing HDL level, and/or decreasing LDL level. In some embodiments,the compositions and methods described herein can be effective to changethe level of lipoproteins and/or lipids in the subject by at least about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, or 60% or even higher as compared to an initial level oflipoproteins and/or lipids prior to administration of it to a subject.The level can be lowered by about 19%-24%, 14%-29%, 12%-35%, 10-40%,8%-45%, 5%-50%, 2%-60%, or 1%-70%. The level can be a circulating level.

In some embodiments, the compositions and methods described herein canbe effective to reduce the atherosclerotic plaque size in a subject byat least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, or 60% or even higher as compared to an initial sizeof atherosclerotic plaque prior to administration of it to a subject.The level can be reduced by about 19%-24%, 14%-29%, 12%-35%, 10-40%,8%-45%, 5%-50%, 2%-60%, or 1%-70%.

Administration of compositions disclosed herein that increase SIRT1 andSIRT3 activity can be useful in any subject in need of metabolicactivation of hepatocytes, adipocytes or one or more of their muscles,e.g., skeletal muscle, smooth muscle or cardiac muscle or muscle cellsthereof. A subject can be a subject having cachexia or muscle wasting.Increasing SIRT3 activity can also be used to increase or maintain bodytemperature, e.g., in hypothermic subjects and increasing SIRT1 activityis beneficial for treating hyperlipidemia, diabetes (type 2 diabetes)and impaired glucose tolerance and reducing inflammatory responses in asubject. Increase in metabolic activation of hepatocytes, adipocytes orone or more of their muscles can be useful in lowering the lipid contentand increasing weight loss of the subject. The content or levels of thelipids and lipoproteins can be lowered.

Increasing SIRT3 activity can also be used for treating or preventinghyperlipidemia, cardiovascular diseases, reducing blood pressure byvasodilation, increasing cardiovascular health, and increasing thecontractile function of vascular tissues, e.g., blood vessels andarteries (e.g., by affecting smooth muscles). Generally, activation ofSIRT3 can be used to stimulate the metabolism of hepatocytes, adipocytesor any type of muscle, e.g., muscles of the gut or digestive system, orthe urinary tract, and thereby can be used to control gut motility,e.g., constipation, and incontinence. SIRT3 activation can also beuseful in erectile dysfunction. It can also be used to stimulate spermmotility, e.g., and be used as a fertility drug. Other embodiments inwhich it would be useful to increase SIRT3 include repair of muscle,such as after a surgery or an accident, increase of muscle mass; andincrease of athletic performance.

Thus the invention provides methods in which beneficial effects areproduced by ingestion of nicotinic acid and/or nicotinamide ribosideand/or any metabolites thereof, along with leucine and/or leucinemetabolites that increase the protein or activity level of SIRT1 orSIRT3. The activity of SIRT1 and SIRT3 can be increased in muscle cellsand/or hepatocytes in the subject. These methods effectively facilitate,increase or stimulate one or more of the following: mimic the benefitsof calorie restriction or exercise in the hepatocyte or muscle cells,increase mitochondrial biogenesis or metabolism, increase mitochondrialactivity and/or endurance in the hepatocytes or muscle cells, sensitizethe muscle cells to insulin, increase fatty acid oxidation in the musclecell, decrease reactive oxygen species (ROS) in the muscle cell,increase PGC-1α and/or UCP3 and/or GLUT4 expression in the hepatocytesor muscle cells, and activate AMP activated protein kinase (AMPK) in thehepatocytes or muscle cells. Various types of muscle cells can becontacted in accordance with the invention. In some embodiments, themuscle cell is a skeletal muscle cell. In certain embodiments, themuscle cell is a cell of a slow-twitch muscle, such as a soleus musclecell.

The compositions can be administered to a subject orally or by any othermethods. Methods of oral administration include administering thecomposition as a liquid, a solid, or a semi-solid that can be taken inthe form of a dietary supplement or a food stuff.

The compositions can be administered periodically. For example, thecompositions can be administered one, two, three, four times a day, oreven more frequent. The subject can be administered every 1, 2, 3, 4, 5,6 or 7 days. In some embodiments, the compositions are administeredthree times daily. The administration can be concurrent with meal timeof a subject. The period of treatment or diet supplementation can be forabout 1, 2, 3, 4, 5, 6, 7, 8, or 9 days, 2 weeks, 1-11 months, or 1year, 2 years, 3, years, 4 years, 5 years or even longer. In someembodiments of the invention, the dosages that are administered to asubject can change or remain constant over the period of treatment. Forexample, the daily dosing amounts can increase or decrease over theperiod of administration.

The length of the period of administration and/or the dosing amounts canbe determined by a physician or any other type of clinician. Thephysician or clinician can observe the subject's response to theadministered compositions and adjust the dosing based on the subject'sperformance. For example, dosing for subjects that show reduced effectsin energy regulation can be increased to achieve desired results.

In some embodiments, the components in the compositions can beadministered together at the same time in the same route, oradministered separately. The components in the compositions can also beadministered subsequently. In some embodiments, leucine and/or leucinemetabolites in the compositions can be administered to a subject inconjunction with nicotinic acid, nicotinamide riboside and/or nicotinicacid metabolites. In some embodiments, the components in thecompositions can be administered at the same or different administrationroute. For example, leucine and/or leucine metabolites can beadministered orally while nicotinic acid, nicotinamide riboside and/ornicotinic acid metabolites can be administered via intravenousinjection. Each of the metabolites can be administered via the same ordifferent administration routes.

In some embodiments, the composition ns administered to a subject can beoptimized for a given subject. For example, the ratio of leucine and/orleucine metabolites to nicotinic acid, nicotinamide riboside and/ornicotinic acid metabolites or the particular components in a combinationcomposition can be adjusted. The ratio and/or particular components canbe selected after evaluation of the subject after being administered oneor more compositions with varying ratios of leucine and/or leucinemetabolites to nicotinic acid, nicotinamide riboside and/or nicotinicacid metabolites or varying combination composition components.

Another aspect of the invention provides for achieving desired effectsin one or more subjects after administration of a combinationcomposition described herein for a specified time period. For example,the beneficial effects of the compositions described herein can beobserved after administration of the compositions to the subject for 1,2, 3, 4, 6, 8, 10, 12, 24, or 52 weeks.

The invention provides for a method of treating subjects, comprisingidentifying a pool of subjects amenable to treatment. The identifyingstep can include one or more screening tests or assays. For example,subjects that are identified as hyperlipidemic, or that have aboveaverage or significantly greater than average body mass indices (BMI)and/or weight can be selected for treatment. The subject can beoverweight or obese, which can be indicated by an above ideal bodyweight of the subject or a BMI that is higher than 25, 30, 40, or 50.The subject can weight more than about 50, 75, 100, 125, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, or 400 lbs. The subjects thathave been on a high fat diet can be selected for treatment as well. Theidentified subjects can then be treated with one or more compositionsdescribed herein. For example, they can be treated with a combinationcomposition comprising nicotinic acid and a branched-chain amino acid.

The invention also provides for methods of manufacturing thecompositions described herein. In some embodiments, the manufacture of acomposition described herein comprises mixing or combining two or morecomponents. These components can include nicotinic acid, nicotinamideriboside and/or nicotinic acid metabolites, and leucine or metabolitesthereof (such as HMB, or KIC). The amount or ratio of components can bethat as described herein. For example, the mass ratio of leucinecompared with resveratrol can be greater than about 80.

In some embodiments, the compositions can be combined or mixed with apharmaceutically active or therapeutic agent, a carrier, and/or anexcipient. Examples of such components are described herein. Thecombined compositions can be formed into a unit dosage as tablets,capsules, gel capsules, slow-release tablets, or the like.

In some embodiments, the composition is prepared such that a solidcomposition containing a substantially homogeneous mixture of the one ormore components is achieved, such that the one or more components aredispersed evenly throughout the composition so that the composition canbe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules.

Kits

The invention also provides kits. The kits include one or morecompositions described herein, in suitable packaging, and can furthercomprise written material that can include instructions for use,discussion of clinical studies, listing of side effects, and the like.Such kits can also include information, such as scientific literaturereferences, package insert materials, clinical trial results, and/orsummaries of these and the like, which indicate or establish theactivities and/or advantages of the composition, and/or which describedosing, administration, side effects, drug interactions, or otherinformation useful to the health care provider. Such information can bebased on the results of various studies, for example, studies usingexperimental animals involving in vivo models and studies based on humanclinical trials. A kit can comprise one or more unit doses describedherein. In some embodiments, a kit comprises about, less than about, ormore than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 30, 31, 60, 90, 120, 150, 180, 210, or more unit doses.Instructions for use can comprise dosing instructions, such asinstructions to take 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unit doses1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times per day. For example, a kitcan comprise a unit dose supplied as a tablet, with each tablet packageseparately, multiples of tablets packaged separately according to thenumber of unit doses per administration (e.g. pairs of tablets), or alltablets packaged together (e.g. in a bottle). As a further example, akit can comprise a unit dose supplied as a bottled drink, the kitcomprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 24, 28, 36,48, 72, or more bottles.

The kit can further contain another agent. In some embodiments, thecompound of the present invention and the agent are provided or packagedas separate compositions in separate containers within the kit. In someembodiments, the compound of the present invention and the agent areprovided or packaged as a single composition within a container in thekit. Suitable packaging and additional articles for use (e.g., measuringcup for liquid preparations, foil wrapping to minimize exposure to air,and the like) are known in the art and can be included in the kit. Kitsdescribed herein can be provided, marketed and/or promoted to healthproviders, including physicians, nurses, pharmacists, formularyofficials, and the like. Kits can also, in some embodiments, be marketeddirectly to the consumer.

In some embodiments, a kit can comprise a multi-day supply of unitdosages. The unit dosages can be any unit dosage described herein. Thekit can comprise instructions directing the administration of themulti-day supply of unit dosages over a period of multiple days. Themulti-day supply can be a one-month supply, a 30-day supply, or amulti-week supply. The multi-day supply can be a 90-day, 180-day,3-month or 6-month supply. The kit can include packaged daily unitdosages, such as packages of 1, 2, 3, 4, or 5 unit dosages. The kit canbe packaged with other dietary supplements, vitamins, and mealreplacement bars, mixes, and beverages.

EXAMPLES Example 1 Effects of Nicotinic Acid/Leucine and NicotinicAcid/Leucine/Resveratrol on Sirt1 Level in Muscle Cells In Vitro

The use of a composition comprising nicotinic acid and leucine asdescribed herein was investigated, wherein the composition comprisesfree leucine and a sub-therapeutic amount of nicotinic acid. Thecomposition activated Sirt1 in the muscle cells and can be used toameliorate a hyperlipidemic condition. A composition further comprisesresveratrol was investigated as well.

C2C12 mouse myoblasts (American Type Culture Collection) were plated ata density of 8000 cells/cm² (10 cm² dish) and grown in Dulbecco'smodified eagle's medium (DMEM) containing 10% fetal bovine serum (FBS),and antibiotics (growth medium) at 37° C. in 5% CO2. For differentiationof C2C12 cells, cells were grown to 100% confluence, transferred todifferentiation medium (DMEM with 2% horse serum and 1%penicillin-streptomycin), and fed with fresh differentiation mediumevery day until myotubes were fully formed (3 days).

A dose-response study was performed by administering the cells withdifferent concentrations of nicotinic acid in order to find thesub-therapeutic amount of nicotinic acid that exerts no effect on thevariable studied. Concentrations of nicotinic acid<100 nM alone werefound to exert no effect, and experimental concentrations were thereforeset below this level, at 10 nM. This sub-therapeutic level of nicotinicacid was then tested in combination with leucine and HMB. The leucineand HMB were at concentrations that have been previously shown to beattainable in diet or supplement while each having no therapeutic effecton these variables when administered alone (0.5 mM for leucine and 5 μMfor HMB).

C2C12 cell myotubes were administered with 10 nM nicotinic acid (NA), 10nM nicotinic acid with 0.5 mM leucine (NA/Leu), 10 nM nicotinic acidwith 0.5 mM leucine and 200 nM resveratrol (NA/R/Leu), 200 nMresveratrol and 0.5 mM leucine (R/Leu), and 10 μM nicotinic acid for 24hours.

Western blotting was performed with SIRT1 antibodies that were obtainedfrom Cell Signaling (Danvers, Mass.). Protein was measured by BCA kit(Thermo Scientific). Total 35 μg of protein from the cell lysate wasresolved on 10% Tris/HCL polyacrylamide gels (Criterion precast gel,Bio-Rad Laboratories, Hercules, Calif.), transferred to PVDF membranes,incubated in blocking buffer (3% BSA in TBS), incubated with primaryantibody, washed and incubated with secondary horseradishperoxidase-conjugated antibody. Visualization and chemiluminescentdetection were conducted using BioRad ChemiDoc instrumentation andsoftware (Bio-Rad Laboratories, Hercules, Calif.). The band intensitywas assessed using Image Lab 4.0 (Bio-Rad Laboratories, Hercules,Calif.), with correction for background and loading controls. Sirt1 wasdetected at 104-115 kDA. Data were analyzed via one-way analysis ofvariance and least significant difference test was used to separatesignificantly different group means.

It was found that nicotinic acid-leucine synergistically stimulatesSirt1 in C2C12 myotubes, with an effect comparable toresveratrol-leucine (FIG. 2, p<0.05). Nicotinic acid alone did not havea significant effect on Sirt1 levels. The three-way combination ofleucine (10 nM)/resveratrol (200 nM) and nicotinic acid (10 nM) exertedmarkedly greater effects, with a 200% increase in Sirt1 levels(p=0.0001).

Example 2 Effects of Nicotinic Acid/Leucine and NicotinicAcid/Leucine/Resveratrol on P-AMPK/AMPK Level in Fat Cells In Vitro

The use of a composition comprising nicotinic acid and leucine asdescribed herein was investigated, wherein the composition comprisesfree leucine and a sub-therapeutic amount of nicotinic acid. Thecomposition increased sirtuin pathway output including AMPK, a signalingmolecule in the sirtuin pathway, and p-AMPK/AMPK level in the fat cellsand can be used to ameliorate a hyperlipidemic condition. A compositionfurther comprises resveratrol was investigated as well.

3T3-L1 preadipocytes (American Type Culture Collection) were plated at adensity of 8000 cells/cm2 (10 cm2 dish) and grown in Dulbecco's modifiedeagle's medium (DMEM) containing 10% fetal bovine serum (FBS), andantibiotics (growth medium) at 37° C. in 5% CO2. Confluent 3T3-L1preadipocytes were induced to differentiate into adipocytes with astandard differentiation medium consisting of DMEM medium supplementedwith 10% FBS, 250 nM dexamethasone, 0.5 mM 3-Isobutyl-1-methylxanthine(IBMX) and 1% penicillin-streptomycin. Preadipocytes were maintained inthis differentiation medium for 3 days and subsequently cultured ingrowth medium. Cultures were re-fed every 2-3 days to allow >90% cellsto reach fully differentiation before conducting chemical treatment.

A dose-response study was performed by administering the cells withdifferent concentrations of nicotinic acid in order to find thesub-therapeutic amount of nicotinic acid that exerts no effect on thevariable studied. Concentrations of nicotinic acid<100 nM alone werefound to exert no effect, and experimental concentrations were thereforeset below this level, at 10 nM. This sub-therapeutic level of nicotinicacid was then tested in combination with leucine and HMB. The leucineand HMB were at concentrations that have been previously shown to beattainable in diet or supplement while each having no therapeutic effecton these variables when administered alone (0.5 mM for leucine and 5 μMfor HMB).

Differentiated 3T3-L1 cells were administered with 10 nM nicotinic acid(NA), 10 nM nicotinic acid with 0.5 mM leucine (NA/Leu), 10 nM nicotinicacid with 0.5 mM leucine and 200 nM resveratrol (NA/R/Leu), 200 nMresveratrol and 0.5 mM leucine (R/Leu), and 10 μM nicotinic acid for 24hours.

Western blotting was performed with antibodies against AMPK andPhospho-AMPKα (Thr172) obtained from Cell Signaling (Danvers, Mass.).Protein was measured by BCA kit (Thermo Scientific). Total 30 μg ofprotein from the cell lysate was resolved on 10% Tris/HCL polyacrylamidegels (Criterion precast gel, Bio-Rad Laboratories, Hercules, Calif.),transferred to PVDF membranes, incubated in blocking buffer (3% BSA inTBS), incubated with primary antibody (P-AMPK), washed and incubatedwith secondary horseradish peroxidase-conjugated antibody. Visualizationand chemiluminescent detection were conducted using BioRad ChemiDocinstrumentation and software (Bio-Rad Laboratories, Hercules, Calif.).The band intensity was assessed using Image Lab 4.0 (Bio-RadLaboratories, Hercules, Calif.), with correction for background andloading controls. AMPK was detected 62 kDA and P-AMPK was detected at64-66 kDA. Data were analyzed via one-way analysis of variance and leastsignificant difference test was used to separate significantly differentgroup means.

It was found that 10 nM nicotinic acid, when administered alone, had nosignificant effect on AMPK activation (FIG. 3). The combination ofnicotinic acid-leucine significantly stimulated AMPK activation tocomparable degree as leucine-resveratrol (p<0.01), as demonstrated by anincrease in P-AMPK/AMPK, while the three-way combination of nicotinicacid-leucine-resveratrol was not significantly different from either ofthe two way leucine combinations (FIG. 2).

Example 3 Effects of Nicotinic Acid/Leucine and NicotinicAcid/Leucine/Resveratrol on Fat Content in C. elegans In Vivo

The use of a composition comprising (a) nicotinic acid and (b) leucineas described herein was investigated, wherein the composition comprisesfree leucine and a sub-therapeutic amount of nicotinic acid. Thecomposition lowered lipid content in a subject after administration ofthe composition to the subject.

Caenorhabditis elegans (C elegans) worms (N2 Bristol wild-type) wereobtained from the Caenorhabditis Genetics Center (CGC) at the Universityof Minnesota and grown on standard NGM plates with E. Coli (OP50) asfood source at 20 degree C. For treatments, eggs were hatched on astarved plate overnight. Then synchronized L1 larvae were transferred toE. coli fed NGM plates containing indicated treatments for about 35hours to reach L4/young adult stage. All treatments were added to theagar. Treatments included 10 nM of nicotinic acid and 0.5 mM of leucine.

Fat content, protein content, fatty acid oxidation of the C elegans wormwere measured using the methods described herein.

For Oil-Red Staining to quantify fat content, treated L4/young adultworms were washed off from plates three times with PBS and collected ina 15 ml conical tube, followed by centrifugation at 1000 g for 30 sec.The supernatant was discarded and the pellet was washed with 10 ml PBS.After centrifugation, the supernatant was discarded except 400 μl, whichwas transferred to a new 1.5 ml eppendorf tube. Then 500 μl of 2×MRWB(160 mM KCl, 40 mM NaCl, 14 mM Na2EGTA, 1 mM Spermidine HCl, 0.4 mMSpermine, 30 mM NaPIPES pH 7.4, 0.2% β-Mercaptoethanol) and 100 μl of20% Paraformaldehyde were added and the samples were gently rocked for60 min at room temperature. Then tubes were centrifuged at 1500 g for 30sec, then aspirated and washed with PBS once, centrifuged again andaspirated to 300 μl. 700 μl of isopropanol was added, mixed by invertingthe tube and incubated with gentle shaking for 15 min at roomtemperature. After centrifuging the tubes to remove the isopropanol, 1ml of 60% filtered Oil-Red-O-dye solution (0.5 g Oil Red 0 in 100 mlanhydrous isopropanol, equilibrated for 2 days by stirring at RT, then 4vol ddH2O was mixed with 6 vol dye solution and equilibrated for 15 minat RT, then filtered with 0.2 M pore size) was added to worms androtated on shaker overnight. Worms were centrifuged at 1200 g for 30sec, and followed by ddH2O washes for 4 times to remove any unboundstain. For quantification, the Oil Red 0 was eluted from the cells byaddition of 100% isopropanol and the optical density of 200 μl aliquots(triplicates/sample) was determined at a wavelength of 540 nm using aBiotek Synergy HT Microplate Reader (BioTek, Winooski, Vt., USA). Datawere normalized to protein content using the Pierce BCA protein assaykit.

To determine the protein content by western blot, treated L4/young adultworms were washed off from plates with M9 buffer and collected intomicrocentrifuge tubes. After centrifugation (500 g for 5 min),supernatant was removed to about 100 μl. Then 250 μl RIPA buffer plusProtease and Phosphatase inhibitor mix was added. Samples werehomogenized, then centrifuged at 16,000 g for 10 min at 40 C. The clearsupernatant was used for further experiments. Protein content wasdetermined using the Pierce BCA protein assay kit.

Fatty acid oxidation was measured by measuring the palmitate-stimulatedoxygen consumption rate with the XF 24 analyzer (Seahorse Bioscience,Billerica, Mass., USA) as previously described (Bruckbauer A, Zemel M B.Synergistic effects of metformin, resveratrol, and hydroxymethylbutyrateon insulin sensitivity. Diabetes, Met Synd Obesity 2013; 6:93-102) withslight modifications. Treated L4/young adult worms were washed off fromplates with M9 buffer and collected into 15 ml conical tubes. Aftercentrifugation (1000 g for 1 min), supernatant was removed and wormpellet was diluted to a concentration of 40 worms/μl. Worms were kept inice water during plating to limit movement, and 5 μl of the wormsolution was added to each well of a 24-well Seahorse islet plates (˜200worms/well). Screens were inserted and 595 μl of M9 buffer withindicated treatments was added to each well. Each plate was cooled for10 min before the start of the measurement. The temperature setting ofthe instrument was maintained at 29 degree C. during the experiment.

Data were analyzed via one-way analysis of variance and leastsignificant difference test was used to separate significantly differentgroup means.

We measured the lipid content in C elegans as shown in FIG. 4. It wasfound that exposing C. elegans to a leucine (0.5 mM)-nicotinic acid (10nM) combination for 24 hours resulted in a 33% decrease in total lipidcontent compared to the non-treated control group.

Example 4 Effects of Nicotinic Acid/Leucine and NicotinicAcid/Leucine/Resveratrol on Triglyceride, LDL, HDL and CholesterolLevels, and Atherosclerotic Plaque Size In Vivo

To assess the efficacy of the subject compounds, mice are administeredthe subject compounds comprising (a) nicotinic acid and/or nicotinamideriboside and/or nicotinic acid metabolites and (b) leucine and/orleucine metabolites as described herein, wherein the compositioncomprises free leucine and a sub-therapeutic amount of nicotinic acid.The composition can be used to lower triglyceride, LDL and cholesterollevels as well as to reduce the size of atherosclerotic plaque in amouse after administration of the composition to the mouse. Acomposition further comprising resveratrol is investigated as well.

LDL receptor knockout (LDLRKO) mice are obtained from JacksonLaboratories (Bar Harbor, Me.), and housed in groups under roomtemperature in a humidity-controlled environment with a regular lightand dark cycle. The mice are provided free access to an atherogenic dietcontaining 0.2% cholesterol (by weight) and 10% calories from saturatedfat (palm oil) and water for 8 weeks prior to treatment. For treatment,the mice are given (a) 250 mg nicotinic acid/kg diet alone, (b) 250 mgof nicotinic acid/kg diet and 24 g of leucine/kg diet, (c) 50 mgnicotinic acid/kg diet, 24 g leucine/kg diet, and 12.5 mg resveratrol/kgdiet, (d) 24 g leucine/kg diet and 12.5 mg resveratrol/kg diet, (e) 10 gnicotinic acid/kg diet alone The treatments are administered for 60 dayscontinuously. Two control groups are included for comparison: thenegative control group receives atherogenic diet with no treatment, anda double-negative control group receives a normal diet with notreatment. All chemicals are purchased from Sigma.

Blood samples of the mice in all groups are obtained from tails of themice at day 7, 30 and 60 after the administration. The serum/plasmalevels of triglyceride, cholesterol, LDL and HDL are measured. Cutaneousvasodilation is measured by laser-Doppler flowmeter. At day 60, mice aresacrificed for quantifying the aortic plaque. Briefly, after dissectionand removal of adherent fat tissue, the aorta is immersed in the abovebuffer overnight. After removal of the adventitia, the aorta is openedlongitudinally, pinned flat onto a dissecting wax, and stained with oilred O for microscopic image analysis. The total aortic area and stainedaortic lesion area are manually outlined in a blinded fashion andanalyzed using Adobe Photoshop CS3, and the lesion size as a percentageof the total aortic area is determined.

Results

Mice receiving only the atherogenic diet with no treatment can exhibitsignificantly higher levels of triglyceride, LDL and cholesterol inbloodstream as compared to the double negative control group receivingnormal diet. For the treatment groups, group (a) can show similar,without statistically significant difference, levels of triglyceride,LDL, cholesterol and HDL as the negative control group receiving onlyatherogenic diet. Groups (b), (c) and (e) may exhibit significantlylower triglyceride, LDL and cholesterol levels, and significantly higherHDL level in the blood stream as compared to the negative control groupreceiving only atherogenic diet, and the levels may decrease (increasefor HDL) over time. Group (d) may exhibit minimal decrease intriglyceride, LDL and cholesterol levels.

For atherosclerotic plaque, groups (b), (c) and (e) may exhibitsignificantly reduced atherosclerotic lesion size in all regions of theaorta. No significant reduction in atherosclerotic lesion size isexpected in groups (a) and (d).

It is also expected that only the mice receiving 10 g of nicotinicacid/kg diet alone exhibit significant higher cutaneous vasodilation ascompared to all the other groups including control groups. The cutaneousvasodilation may be found lower in the groups (a) to (d) as compared togroup (e).

Overall, nicotinic acid with a dose that is 250 mg/kg diet administeredin conjunction with 24 g leucine/kg diet may exhibit similar effects onlowering the triglyceride, LDL, and cholesterol level as well asincreasing the HDL level in mice as compared to 10 g nicotinic acid/kgdiet alone without increasing the cutaneous vasodilation significantly.Low dose of nicotinic acid administered in conjunction with leucine andresveratrol is expected to exhibit similar effects.

Example 5 Effects of Nicotinic Acid/Leucine and NicotinicAcid/Leucine/Resveratrol on Triglyceride, LDL, HDL and CholesterolLevels in Human

To assess the efficacy of the subject compounds, humans are administeredthe subject compounds comprising (a) nicotinic acid and/or nicotinamideriboside and/or nicotinic acid metabolites and (b) leucine and/orleucine metabolites as described herein, wherein the compositioncomprises free leucine and a sub-therapeutic amount of nicotinic acid.The composition can be used to lower triglyceride, LDL and cholesterollevels in a human after administering the composition to the human. Acomposition further comprising resveratrol is investigated as well.

Patients that are pre-diagnosed with hyperlipidemia are admitted for therandomized and double blind study. Each patient is administered orally(a) 50 mg nicotinic acid alone, (b) 50 mg of nicotinic acid and 1135 mgof leucine, (c) 50 mg nicotinic acid, 1135 mg leucine, and 50 mgresveratrol, (d) 1135 mg leucine and 50 mg resveratrol, (e) 3000 mgnicotinic acid alone, and (f) placebo. The treatments are administeredorally twice a day, for 60 days continuously.

Blood samples of the patients in all groups are obtained from at day 0,30 and 60 after the administration. The serum/plasma levels oftriglyceride, cholesterol, LDL and HDL are measured. Cutaneousvasodilation is measured by laser-Doppler flowmeter and the discomfortlevel described by the patients.

Results

For the treatment groups, group (a) and (f) may show similar, withoutstatistically significant difference, levels of triglyceride, LDL,cholesterol and HDL as compared to day 0 values. Groups (b), (c) and (e)may exhibit significantly lower triglyceride, LDL and cholesterollevels, and significantly higher HDL level in the blood stream ascompared to the respective day 0 values. Group (d) may exhibit minimaldecrease in triglyceride, LDL and cholesterol levels.

It is also expected that only the patients receiving 3000 mg ofnicotinic acid alone exhibit significant higher cutaneous vasodilationand more complaints from the patients as compared to all the othergroups including placebo. The cutaneous vasodilation may be lower in thegroups (a) to (d) as compared to group (e).

Overall, nicotinic acid with a dose that is 50 mg administered inconjunction with 1135 mg leucine may exhibit similar effects on loweringthe triglyceride, LDL, and cholesterol level as well as increasing theHDL level in patients as compared to 3 g nicotinic acid alone withoutincreasing the cutaneous vasodilation significantly. 50 mg of nicotinicacid+1135 mg leucine administered in conjunction with 50 mg resveratrolmay exhibit similar effects.

Example 6 Effects of Nicotinic Acid/Leucine and NicotinicAcid/Leucine/Resveratrol on Atherosclerotic Plaque Size in Human

To assess the efficacy of the subject compounds, humans are administeredthe subject compounds as described herein, wherein the compositioncomprises free leucine and a sub-therapeutic amount of nicotinic acid.The composition can be used to reduce the size of atherosclerotic plaquein a human after administering the composition to the human. Acomposition further comprising resveratrol is investigated as well.

Patients that experience acute chest pain and are pre-diagnosed withhyperlipidemia are admitted for the randomized and double blind study.Each patient is administered orally (a) 50 mg nicotinic acid alone, (b)50 mg of nicotinic acid and 1135 mg of leucine, (c) 50 mg nicotinicacid, 1135 mg leucine, and 50 mg resveratrol, (d) 1135 mg leucine and 50mg resveratrol, and (e) placebo. The treatments are administered orallytwice a day, for 3 years continuously. The size of atheroscleroticplaque is measured at day 0, months 6, 12, 18, 24, 30 and 36 byquantitative coronary angiography.

Results

For the treatment groups, group (a) and (e) may show similar, withoutstatistically significant difference, size of atherosclerotic lesion ascompared to day 0 values. Groups (b) and (c) may exhibit significantlyreduced atherosclerotic plaque size as compared to the respective day 0values. Group (d) may exhibit minimal decrease in atherosclerotic plaquesize.

Example 7 Effects of Nicotinic Acid/Leucine on Triglyceride, LDL, HDLand Cholesterol Levels In Vivo

To assess the efficacy of the subject compounds, mice were administeredthe subject compounds comprising (a) nicotinic acid and (b) leucine asdescribed herein, wherein the composition comprises free leucine and asub-therapeutic amount of nicotinic acid. The composition lowered thetriglyceride, LDL and cholesterol levels in a mouse after administrationof the composition to the mouse.

LDL receptor knockout (LDLRKO) mice were obtained from JacksonLaboratories (Bar Harbor, Me.), and housed in groups under roomtemperature in a humidity-controlled environment with a regular lightand dark cycle. The mice were provided free access to an atherogenicwestern diet (WD) containing 0.21% cholesterol (by weight) and 40%calories from fat and water for 4 weeks prior to treatment. Fortreatment, the mice were given (a) the WD diet alone, (b) WD and 24 g ofleucine/kg diet, (c) WD and 24 g of leucine/kg diet and 50 mg nicotinicacid/kg diet, (d) WD and 24 g of leucine/kg diet and 250 mg nicotinicacid/kg diet, or (e) WD and 1000 mg nicotinic acid/kg diet; this isapproximately equivalent to a low therapeutic dose of nicotinic acid inhypercholesterolemic humans (˜1,500 mg/day). The treatments wereadministered for 28 days continuously.

Blood samples of the mice in all groups were obtained from tails of themice at day 28 after the administration. The serum/plasma levels oftriglyceride, total cholesterol and cholesterol esters were measured.

Results

The atherogenic western diet resulted in profound elevations in plasmacholesterol (FIG. 5), cholesterol esters (FIG. 6), and trigylcyerides(FIG. 7). Addition of a therapeutic dose of nicotinic acid (1,000 mg/kgdiet) resulted in a 20% decrease in total cholesterol (p<0.01, FIG. 5).Although leucine exerted no independent effect on total cholesterol,addition of leucine to sub-therapeutic doses of nicotinic acid (50 or250 mg/kg diet) resulted in comparable decreases in total cholesterol tothat found with the therapeutic dose (p<0.01, FIG. 5). Similarly,addition of a therapeutic dose of nicotinic acid (1,000 mg/kg diet)resulted in a 28% decrease in cholesterol esters (p<0.002, FIG. 6), anda statistically comparable decrease was found when leucine was added tosub-therapeutic doses of nicotinic acid (50 or 250 mg/kg diet)(p<0.002,FIG. 6). Leucine exerted no independent effect on cholesterol esters.Plasma triglycerides were similarly affected. Addition of a therapeuticdose of nicotinic acid (1,000 mg/kg diet) resulted in a 32% decrease inplasma triglycerides (p<0.01, FIG. 7), and a statistically comparabledecrease in triglycerides was found when leucine was added tosub-therapeutic doses of nicotinic acid (50 or 250 mg/kg diet) (p<0.01,FIG. 7).

Example 8 Effects of Leucine-Nicotinic Acid on the Lifespan in C.elegans

The use of a composition comprising (a) nicotinic acid and (b) leucineas described herein was investigated, wherein the composition comprisesfree leucine and a sub-therapeutic amount of nicotinic acid. Thecomposition synergistically extended the lifespan in a subject afteradministration of the composition to the subject.

Worms (N2 Bristol wild-type) were obtained from the CaenorhabditisGenetics Center (CGC) at the University of Minnesota and grown onstandard NGM plates with E. coli (OP50) as food source at 20° C. Eggswere hatched on a starved plate overnight. Then synchronized L1 larvaewere transferred to E. coli fed NGM plates containing indicatedtreatments for about 35 hours to reach L4/young adult stage. To studylifespan, 50 young adult worms were placed on NGM agar plates seededwith E. coli strain OP-50 (=day 1 of study). All treatments were addedwith the indicated concentrations to the agar plates. Treatmentsincluded 10 nM of nicotinic acid and 0.5 mM of leucine.

The worms were maintained at 21° C. throughout the duration of thestudy. Worms were transferred to new plates daily to eliminate progeny.Worms were scored as dead if they did not respond to repeated touch witha platinum pick. The study was continued until the last animal was dead.Data were analyzed via Kaplan-Meier survival curves using Prism 6(GraphPad Software) and statistical significance was determined by theLog-rank (Mantel-Cox) test.

It was found that leucine (0.5 mM) and nicotinic acid (10 nM) eachexerted no independent effect on lifespan, but when combined extendedmaximal lifespan under basal conditions, and extended median lifespan by28% under conditions of oxidative stress induced by administration ofparaquat (0.2 mM) (FIG. 8).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein can be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A composition comprising: a) at least 250 mg ofleucine and/or at least 25 mg of one or more leucine metabolites,wherein the leucine metabolite is selected from the group consisting ofketo-isocaproic acid (KIC), alpha-hydroxy-isocaproic acid, andhydroxymethylbutyrate (HMB); and b) at least 1 mg of nicotinic acidand/or nicotinamide riboside and/or one or more nicotinic acidmetabolites, wherein the nicotinic acid metabolite is selected from thegroup consisting of nicotinyl CoA, nicotinuric acid, nicotinatemononucleotide, nicotinate adenine dinucleotide, and nicotinamideadenine dinucleotide; and c) an anti-hyperlipidemic agent; wherein thecomposition is substantially free of alanine, arginine, asparagine,aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, valine, isoleucine and tyrosine.
 2. The composition of claim1, wherein the composition is substantially free of nicotinamide.
 3. Thecomposition of claim 1, wherein the component (a) in the composition isleucine and the component (b) in the composition is nicotinic acid. 4.The composition of claim 1, wherein the amount of leucine and/or one ormore leucine metabolites is less than 1300 mg.
 5. The composition ofclaim 1, wherein the amount of nicotinic acid and/or nicotinamideriboside and/or one or more nicotinic acid metabolites is less than 250mg.
 6. The composition of claim 1, wherein the amount of nicotinic acidand/or nicotinamide riboside and/or one or more nicotinic acidmetabolites is about 1-100 mg.
 7. The composition of claim 1, whereinthe composition further comprises resveratrol.
 8. The composition ofclaim 1, wherein the composition is a tablet, a capsule, or a pill. 9.The composition of claim 1, wherein the composition contains less than10% of non-leucine amino acids.
 10. The composition of claim 1, whereinthe anti-hyperlipidemic agent is selected from the group consisting ofHMG-CoA inhibitor, fibrate, bile acid sequestrant, ezetimibe,lomitapide, phytosterols, CETP antagonist, orlistat, dextrothyroxinesodium, icosapent, and any combination thereof.
 11. The composition ofclaim 1, wherein the anti-hyperlipidemic agent comprises a HMC-Co Ainhibitor selected from the group consisting of atorvastatin,fluvastatin, pravastatin, lovastatin, simvastatin, pitavastatin,cerivastatin, rosuvastatin, and lovastatin/niacin ER.
 12. Thecomposition of claim 1, wherein the anti-hyperlipidemic agent comprisessimvastatin and ezetimibe.
 13. The composition of claim 1, wherein theanti-hyperlipidemic agent comprises a fibrate selected from the groupconsisting of gemifibrozil, fenofibrate, fenofibric acid, clofibrate,and micronized fenofibrate.
 14. The composition of claim 1, wherein theanti-hyperlipidemic agent comprises a bile acid sequestrant selectedfrom the group consisting of colestipol, cholestyramine and colesevelam.15. The composition of claim 1, wherein the composition is formulated ina unit dosage form.
 16. The composition of claim 1, wherein a portion ofthe leucine and/or one or more leucine metabolites is in a free form.17. The composition of claim 1, wherein the composition whenadministered to a subject in need thereof synergistically reduces lipidcontent in the subject.
 18. The composition of claim 1, wherein thecomposition when administered to a subject in need thereofsynergistically enhances a decrease in weight gain, enhances an increasein fat oxidation, and enhances an increase in activation of Sirt1 in thesubject.
 19. A method of lowering total cholesterol level or total lipidcontent in a subject in need thereof, comprising administering to thesubject a composition of claim 1 to effect a reduction in the totalcholesterol level or total lipid content in the subject.
 20. A method oflowering triglyceride level, total cholesterol or LDL level in a subjectin need thereof, comprising administering to the subject a compositionof claim 1 to effect a reduction in the triglyceride level, totalcholesterol or LDL level in the subject.